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Three‐spot seahorse (Hippocampus trimaculatus) has been consumed as traditional Chinese medicine in Asian society. This study was designed to analyze the bioactive compounds of the solvent extracts from cultured three‐spot seahorse by high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC‐ESI/MS/MS). Subsequently, their biological activities were evaluated and confirmed by cell modes and Western blot analysis. Experimental results indicated that taurine and arginine were the primary bioactive compounds identified and quantified without pre‐ or post‐column derivatization within 20 min retention time. The analytical method was established and validated with intraday/interday RSD from 0.25% to 3.34% and with recovery from 87.8% to 91.2%. As compared to other extracts, water layer extract (WLE) contained the most taurine and arginine contents of 6.807 and 0.437 mg/g (dry basis), respectively. In the meanwhile, WLE also showed anti‐inflammatory activity on LPS‐induced NO production and inhibited the protein expression of TNF‐α and COX‐2 by Western blot analysis with better cell viability. [ABSTRACT FROM AUTHOR]