For quality control purpose, an approach of chemical fingerprinting of Liuwei Dihuang Pill (LDP) and simultaneous determination of its multiple bioactive components were established by using high performance liquid chromatograph (HPLC) coupled with multiple detection techniques. HPLC with ultraviolet detection (HPLC-UV) was used to acquire its fingerprint, and HPLC with combined detections of diode array detector and evaporative light scattering detector (HPLC-DAD-ELSD) was performed to simultaneously determine eight bioactive constituents: including gallic acid, 5-hydroxymethyl furfural, morroniside, sweroside, loganin, paeoniflorin, paeonol and alisol B-23 acetate. The detection limits and quantification limits ranged in 0.11-1.93mug/mL and 0.38-3.85mug/mL, respectively. The validation of the proposed approach was acceptable, with 93.47-104.62% accuracy in recovery test. The intra- and inter-day precisions of the method were evaluated and were less than 3.87%, with accuracy from 95.3% to 103.4%. In addition, the mass spectrometry of the investigated major constituents was also studied. Based on the chromatographic fingerprint data, partial least square (PLS) and discriminate analysis were utilized to visualize the quality information of 60 batches of LDP, and a partial least square-discriminate analysis (PLS-DA) model was constructed with acceptable predictive performance for the discrimination of various products. The proposed approach was expected to be developed as a powerful tool for the quality control of LDP.