Introduction: Early pregnancy diagnosis is a key factor in shortening the calving interval through identifying the non-pregnant animals to rebreed them at the earliest time after artificial insemination (AI). Bovine pregnancy-associated glycoproteins (PAGs) are produced by mono-and binucleated trophoblastic cells of the pregnant cow’ s placenta. Detection of PAG in the maternal circulation has been used to accurately diagnose pregnancy. Several studies have used commercial PAG tests to determine pregnancy status in dairy cows and heifers. Pregnancy-specific protein B (PSPB) or PAG1 was the first identified member of the PAG family and commercial diagnostic kits still utilize PAG1 as a pregnancy marker. In recent years, the use of nanoparticles in immunosensors has been increased their sensitivity and increased the traceability of antigenantibody responses. Due to the limitation of using ultrasound and other methods for pregnancy diagnosis in the first 30 days after inoculation, the aim of this study was to apply the pregnancy-associated glycoprotein detection method in order to reduce the time of pregnancy diagnosis. For this purpose, a sandwich ELISA immunosensor was designed for the detection of PAGs and named Nano-kit. Materials and Methods: Magnetic nanoparticles were used to enhance the contact area between antibodies and antigens. Streptavidin and biotin were used for their high binding affinity to bind the antibodies and enzymes to nanomagnet. The synchronization technique and artificial insemination (AI) was performed in Holstein dairy cows (n = 58). Pregnancy status was determined by transrectal ultrasonography (30 days after AI). Furthermore, transrectal palpation was carried out by a skilled veterinarian on day 60 after AI to determine the pregnancy status of cows which were previously detected as pregnant. For PAG1 analysis, blood samples (10 mL) were collected daily from 18 to 30 days after AI. Blood samples were collected by venipuncture from the tail vein into evacuated tubes containing EDTA as an anticoagulant and were processed 1– 3 h after sampling. The samples were centrifuged and plasma samples were transferred to fresh tubes and were stored at − 20° C until they were assessed. Plasma concentrations of PAG1 were determined by nano-kit and commercial kit. Differences in the level of PAG-1 were evaluated by repeated measures ANOVA (SAS 9. 4) over days of sampling (18 to 30). Values of PAG-1 in pregnant cows were considered as the reference. Data were presented as means ± SD and differences were considered significant at P<0. 05. Results and Discussion: By doing Ovsynch protocol, of the 54 cows enrolled in the first AI, 48% (26/54) of synchronized cows were diagnosed pregnant 30 days after AI using transrectal ultrasonography. From day 30 to 60 after AI, 73% (19/26) of cows maintained pregnant and the pregnancy loss from day 32 to 60 after AI was 17% (7/26). Measurement of different concentrations of PAG1 (standards) using a commercial kit and nano-kit showed that nano-kit were more sensitive than the commercial kit and detected a concentration of 0. 03 ng/mL. The first increase in plasma concentration of PAG1 occurred on d 23 after AI in pregnant cows and PAG1 concentration in serum increased from d 22 to 30 after AI and it was affected by day (P < 0. 001). Cows diagnosed as pregnant on day 60 after AI had a higher PAG concentration on day 24 compared with cows that were diagnosed as non-pregnant (2. 28 ± 0. 15 ng/mL vs 0. 7 ± 0. 18 ng/mL, respectively; P < 0. 001). Accuracy in predicting pregnancy at day 24 of gestation based on circulating concentration of PAGs was 95 % for 1. 93 ng/mL. Generally, based on the results of this study, the cows with mean PAG1 concentration more than 2. 28 ± 0. 07 ng/mL on 24 and mean PAG1 concentration more than 9. 2 ± 0. 07 ng/mL on day 30 of pregnancy, remained pregnant until day 60 of pregnancy. Furthermore, in cows diagnosed pregnant on day 24 of pregnancy but were not pregnant on day 30 after AI, blood plasma PAG1 concentration on day 24 after AI was 1. 03 ± 0. 66 ng/ml. Conclusion: Based on these results, PAG concentrations at day 24 of gestation are higher in pregnant compared to non-pregnant dairy cows and could be applied in diagnosing pregnancy at day 24 of gestation; however, further study is needed to determine the potential of PAG1 in pregnancy diagnosis in dairy cows.