Targeting isolation of biflavonoids and selaginellin derivatives from Selaginella pulvinata (Hook.& Grev.) Maxim. was investagated by was performed on a Waters preparative LC-MS autopurification system and the ethyl acetate part of the 95% ethanol extract of S. pulvinata was separated and purified. The chemical structures of the separated compounds were identified by MS,¹H NMR,¹³C NMR,2D NMR and spectral analysis methods. Four biflavonoids were isolated and identified as hinokiflavone (1),isocryptomerin (2),sotetsuflavone (3) and amentoflavone (4). Seven selaginellin derivatives were obtained and identified as selaginellin (5),selaginpulvilin A (6),selaginpulvilin B (7),selaginellin O (8),selaginellin E (9),selaginellin A (10) and selaginellin B (11). The MTT assay was used to evaluate the inhibitory effect of compounds 1~11 on the proliferation of human non-small cell lung cancer cell line (NSCLC) H322 in vitro. The results showed that compounds 1,2,3,5,6,8,9,10 and 11 had different degrees of inhibitory activity on human NSCLC cell line H322. Compounds 3 and 10 had stronger inhibitory activity on human NSCLC cell line H322,with IC₅₀ values of 16.70 μmol/L and 9.54 μmol/L,respectively.