In situ synthesis of layered double hydroxides (LDHs) is an effective way to extract short chain fatty acids (SCFAs) from anaerobic fermentation liquid to be used as carbon source for biodenitrification, but the slow release performance need to be improved. Three layered double hydroxides with different metal cations (Mg-Al, Ni-Al, Ni-Fe) intercalated with acetate (Ac) were successfully prepared in the present study. The release rates decreased in the order of MgAl-LDH > NiAl-LDH > NiFe-LDH, meaning that NiFe-LDH-Ac showed the best slow release performance. The release rate of LDHs decreased greatly along with the increase of particle size. Batch denitrification tests showed that the carbon source utilization efficiency of NiFe-LDH-Ac was 79.9%, which was much higher than that of CH3COONa (48.8%) as control. These results indicate that NiFe-LDH-Ac could be successfully employed as slow release carbon source for biodenitrification.