Objective To verify the methodology of electrochemical luminescence immunoassay (ECLIA) for determination of 24 hours urinary free cortisol. Methods The 24 hours urinary free cortisol levels in 24 in-patients were determined by using ECLIA and CLIA extraction methods respectively. The 24 hours urine of 87 health controls were collected and the free cortisol levels were measured with ECLIA. The imprecision, function sensitivity and analytical measurement range of ECLIA were verified and reference range was established. Results There were positively correlations between ECLIA and CLIA exactions in the determination of 24 hours urinary free cortisol (r=0.9724, P<0.001). The high and low values of within-run imprecision of ECLIA were 3.30% and 4.52%, and between-day imprecision were 4.69% and 6.55% respectively. The analytical measurement range was 0.053~58.91μg/dL (r2=0.9882, y=1.0344x+0.8466) and the function sensitivity was 0.05 μg/dL. The reference range of 24 hours urinary free cortisol was 30.1-129.1 μg. Conclusion The electrochemical luminescence immunoassay of urinary free cortisol has appropriate imprecision, and it could indicate the secretion of 24 hours free cortisol in urine.