Okra is a kind of flavonoid-rich food which was reported to have a variety of health functions. Flavonoids are the major polyphenolic compounds in okra and are thought to play a role in reducing the risk of disease. The aim of this study was to isolate and identify the flavonoids composition in okra pods and explore the activity of the main flavonoids components identified on inhibiting tumor cell proliferation in vitro. Six individual flavonoids were identified by HPLC-MS/MS: quercetin-3-gentiobioside (Q3G), quercetin-3-sambubioside (Q3S), rutin, quercetin-7-glucoside (Q7G), isoquercitrin (ISO) and quercetin-3-malonylglucoside (Q3M), which were all separated well within 30 min. The analytical method was validated by the recovery of spiked samples and so on. Moreover, four main flavonoids components, namely Q3G, Q3S, ISO and Q3M, exhibited significant (p < 0.05) inhibition of NCI-N87, A375, A549 cells proliferation (25−100 μmol/L) and of HFLS-RA (200−300 μmol/L) in different levels, according to MTT method, respectively. It is demonstrated that the flavonoids components of okra exhibited a noteworthy development prospect as a possible nutraceutical dietary supplement.