Objective To study the effect of creatinine, triglyceride, cholesterol and high-density lipoprotein reagents on determination results of serum total bile acid. Methods WTBZTBA content of 30 serum samples(mixed serum) was independently detected. The impact of CR, TC, TG, and HDL reagents on detection of TBA was investigated and detected again after special cleaning procedures. Results There was clear cross-contamination in detection of TBA using CR, TC, TG, HDL reagents. Conclusions Rational detection order and special testing and cleaning procedures can effectively eliminate the effect of CR, TC, TG, and HDL reagents on TBA examination.