Objective To prepare a doxycycline thermo-sensitive in situ gel uterine perfusate, evaluate its quality, and monitor drug concentration in sow uterus.Method Poloxamer 407 (P407) and poloxamer 188 (P188) were used as the main thermo-sensitive materials. The in situ gel was prepared by “cold” process. The prescription was screened and optimized by single factor analysis and orthogonal experiment. The in vitro dissolution and drug release of the preparation were measured by membrane free dissolution model. The stability of the developed thermo-sensitive gel was investigated under the conditions of high temperature, high humidity and strong light. Five binary hybrid sows were selected to monitor drug concentration in sow uterus.Result The final optimized prescription was doxycycline 1 g, P407 10 g, P188 2.5 g, magnesium chloride 2.3 g and sodium bisulfite 0.1 g in per 50 g preparation, pH was adjusted to 4.0±0.2 with ethanolamine, and the residue was ultrapure water. The in vitro dissolution and drug release time of the preparation lasted for 48 h, the cumulative dissolution rate and drug release rate were up to 100%. The preparation stability was complied with technical guidelines for the stability of veterinary chemical drugs. The drug concentration remained at 68.54 μg/mL after 96 h of the second administration.Conclusion The doxycycline thermo-sensitive in situ gel uterine perfusate is a simple preparation method and shows good stability. The local concentration of the drug in sow uterus maintains at a high level for a long time. The preparation has a sustained release effect and is expected to be an innovative way to treat sow endometritis.