Strong anion exchange (SAX) columns and oasis HLB cartridges were combined to extract trace level oxytetracycline(OTC) and high performance liquid chromatography(HPLC) UV-detector were used to measure it in wastewater sample. The effect of mobile phase was investigated. Under the best analysis conditions, linear calibration equations were established for OTC. The linearity were in the range of 0.1-10g/mL, Correlation coefficient (R2) is about 0.9997. The recovery factors ranged from 82-92% with a relative standard deviation of 4.8-6.4%.Lab-scale experiments were conducted to investigate the sorption behavior of OTC by anaerobic sludge. Starch in the mixed liquor can decrease the adsorption capacity of OTC.