Nowadays, by increasing consumer awareness about the hazards of chemical preservatives, the demand for foods with increased natural preservatives has been enhanced. The main objective of this study was to replace annatto and red rice with nitrite in sausage. For this purpose, annatto replaced by zero, 25, 50, 75 and 100% of nitrite then the sausage samples were made with 60% red meat and 0.05% fixed amount of red rice. Chemical, microbiological and sensory evaluations were performed on days 0, 7, 14, 21 and 28 of storage. The results of chemical and microbial tests showed that the annatto and red rice did not show negative effects on sausage with 60% meat. Based on microbial assay, the total microbial population in group A (0% annatto, 100% nitrite), group B (25% annatto, 75% nitrite), group C (50% annatto, 50% nitrite) and group D (75% annatto, 25% nitrite) was decreased; however, in group E (100% annatto, 0% nitrite) no bacterial decline was observed. Moreover, Clostridium perfringens was not detected in any of the samples. According to the results of sensory evaluations, group C and D had the highest satisfaction in terms of color, taste, odor, consistency and overall acceptance. Meanwhile, a significant difference was observed between all sensory factors in group E and the rest of groups. Considering the quality characteristics, the group containing 75% annatto had the highest approval in comparison with the other samples during the storage period.