Background and purpose: Today, due to the reduction of water resources, separation of graywater from domestic wastewater and its reuse has gained more interest. The aim of this study was toapply multi-layer filtration (MLF) for removal of nutrients from gray water. Materials and methods: This laboratory-scale study was carried out in 2. 3-19. 2 gr. COD / L. dorganic loading rates (OLR) over a period of 157 days. In this study, synthetic gray water was used asinput to the multi-layer filter. The MLF system included three layers: sand, silica, and granular activatedcarbon. Furthermore, the scanning electron microscopy (SEM) images were used to determine the biofilmformation on the media surface. Results: The best removal efficiencies of COD, BOD5, TKN, and TP were 98. 87, 99. 84, 99. 5, and 98% achieved in OLR 19. 2, 12. 8, (13. 2, 4. 6) gCOD/l. d, SOR 5. 76, 2. 88, and 5. 77 m/hr, respectively. The study on the effect of loading rate revealed that by increase in OLR, the system efficiency to removeBOD5 and COD increased. Nevertheless, for TKN and TP, the efficiency decreased with increase in OLR. Also, according to SEM images, biofilm had grown well in the media, which can be effective in removingpollutants from gray water. Conclusion: Using multilayer filtration could be an appropriate option in removing nutrientsfrom gray water in different loadings.