The occurrence and distribution of four selected antibiotics, include tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC) and doxycycline (DOC), in organic fertilizers were investigated at three typical protected vegetable fields in Guli, Suoshi and Donglu villages, suburb of Nanjing City. These four antibiotics were extracted by solvent, and detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The results showed that TCs antibiotics were detectable in all the organic fertilizer samples. The highest concentration was OTC, TC and DOC were the second, and CTC had a lowest level. In those three different regions of Guli, Suoshi and Donglu, the concentration of which ranged from 126–8 071, 266–3 326, 339–373 μg/kg, and with the average of 2 152, 1 188, 356 μg/kg. The results also suggested that the concentration of antibiotics in different types of organic fertilizers were quite variable, especially the total concentrations of animal and human excreta, ranged from 371 to 7 820 μg/kg. Therefore, tetracyclines pollution from organic fertilizers might threaten to the soil environment, and need be concerned.