Aims: The purpose of this paper was to develop a HPLC fingerprint analysis method, compare the chromatographic fingerprints characteristics of the ethyl acetate extract with anticancer activity from Sedi Linearis Herba (Sedum lineare Thumb.) collected in different seasons and environments, determine the contents of active ingredients hyperoside, isoquercetin and astragalin of each sample by HPLC, provide believable scientific foundations for quality control and evaluation of Sedi Linearis Herba. Methods: The analyte was analyzed using BDS Hypersil C18 as chromatographic column, acetonitrile -0.1% acetic acid solution as the mobile phase (gradient elution). Results: The results showed that the major active ingredient contents of the samples collected in different seasons but at the same environment all had obvious differences. These ingredient contents of the samples collected in the end of April were much more than those in mid-August in general. Moreover, the major active ingredient contents of the samples picked in the same season but different environments were different too. For instance, the major ingredient contents of the sample in damp environment in August were more than those in sunny environment. Conclusion: All of these indicated that the HPLC fingerprint analysis and contents determination method established were very useful for quality analysis and control of Sedi Linearis Herba.