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Rationale Quantitative analysis of cholesterol and its metabolic steroid hormones plays a vital role in diagnosing endocrine disorders and understanding disease progression, as well as in clinical medicine studies. Because of their extremely low abundance in body fluids, it remains a challenging task to develop a sensitive detection method. Methods A hyphenated technique of dual ultrasonic-assisted dispersive liquid–liquid microextraction (dual-UADLLME) coupled with microwave-assisted derivatization (MAD) was proposed for cleansing, enrichment and sensitivity enhancement. 4′-Carboxy-substituted rosamine (CSR) was synthesized and used as derivatization reagent. An ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method was developed for determination of cholesterol and its metabolic steroid hormones in the multiple reaction monitoring mode. Results Parameters of dual-UADLLME, MAD and UHPLC–MS/MS were all optimized. Satisfactory linearity, recovery, repeatability, accuracy and precision, absence of matrix effect and extremely low limits of detection (LODs, 0.08–0.15 pg mL−1) were achieved. Conclusions Through the combination of dual-UADLLME and MAD, a determination method for cholesterol and its metabolic steroid hormones in human plasma, serum and urine samples was developed and validated with high sensitivity, selectivity, accuracy and perfect matrix effect results. Copyright © 2016 John Wiley & Sons, Ltd.