Objective To ascertain the best ratio of Tramadol compound injection. Methods The best analgesic effect in different ratios of drugs was selected by orthogonal design method. The analgesic effects in different ratios of drugs were determined by acetic acid-induced writhing and tail-flicking in mice. Results The best analgesic effect in Tramadol compound injection was in the ratio of A2B3. Conclusion The analgesic effect of Tramadol compound injection have the best ratio and cooperative effect.