To optimize the in-situ gel formulation of andrographolide nanoemulsion using the Box-Behnken response surface methodology to provide references for the research and development of transdermal drug delivery systems of Chinese medicine.Methods:A quadratic regression equation was obtained through polynomial fitting analysis with poloxamer 188(P188) and poloxamer 407(P407) as gel matrices，the mass fractions of gel matrices as the investigation factors，and gel temperature as the investigation index，and the mathematical relationship between gel temperature and the two factors were established.The Box-Behnken design-response surface methodology was used to optimize the in-situ gel formulation of andrographolide nanoemulsion，and the optimized formulation was validated.Results:There was a reliable quantitative relationship between gel temperature and the investigation factors，and the quadratic regression equation had a high degree of confidence.The optimal formulation for andrographolide nanoemulsion in-situ gel was determined as 20.00% P407 and 5.00% P188，with a gel temperature of(33.8±0.2)℃.The formulation preparation showed high reproducibility.Conclusion:This method is suitable for optimizing the formulation of andrographolide nanoemulsion in-situ gel.The established model has good predictive performance，and the optimized preparation method is stable with an appropriate gel temperature.This research provides references for the development of andrographolide transdermal drug delivery systems.