This paper describes a sensitive and convenient method to separate progesterone, 17alpha-hydroxy progesterone, cortexolone, hydrocortisone and cortisone, all of which are steroids and have similar structures, using microfluidic chip-based technology with UV detection at 252 nm. We successfully obtained high-speed separation of the five steroids within 70 s in optimized microfluidic controls and micellar electrokinetic chromatography (MEKC) separation conditions. Fairly good linearity with correlation coefficient of over 0.98 from 10 or 20 to 100 mg/l steroid chemicals was obtained. The limits of detection obtained at a signal to noise ratio of 3 were from 3.89 to 7.80 mg/l. The values of the relative standard deviation (RSD) were 0.98-1.34% for repetitive injection (n = 12) and the intraday and interday RSDs were below 6%. The highly stable response reflected the feasibility of this method.