To investigate the anti-human epidermal squamous-cell carcinoma A431 cell proliferative fraction of Euphorbia fischeriana Steud. and to determine chamaejasmine B content in the fraction. Anti-A431 cell proliferative activity of various Euphorbia fischeriana Steud. fractions, which were extracted using ethanol extraction and with different solvents, were screened by MTT assay to identify the active fraction. Chamaejasmine B content in the active fraction was determined by HPLC. Ethyl ether fraction showed more pronounced anti-proliferation activity on A431 cells. Ethyl ether fraction was the active fraction, in which chamaejasmine b content was 30.6%. IC50 of ethyl ether fraction of Euphorbia fischeriana Steud. for A43l cells at 48 h was 40 μmol/L. At this dose, A431 cell growth inhibition rate was (53.2 ± 0.31%). Ethyl ether extract is the anti-A431 cell proliferative fraction of Euphorbia fischeriana Steud. HPLC determination of chamaejasmine B content in active fraction is an accurate, reproducible and convenient method. We preliminarily conclude that the higher the chamaejasmine B content in the active fraction, the stronger its anti-proliferative effect on A431 cells.