Objective To study the chemical constituents of Hippocampus trimaculatus Leach. Methods After extracted with ethanol, Hippocampus trimaculatus Leach was isolated and purified by silica gel column chromatography, Sephadex LH-20 gel column chromatography, and reversed-phase C₁₈ column chromatography. The structures of compounds were identified by physical and chemical properties, spectral data and literature comparison. Results Eight compounds were isolated from Hippocampus trimaculatus Leach and identified as L-phenylalanine (1), alanine (2), inosine (3), cholesterol (4), N-acetyltyramine (5), uracil (6), D-mannitol (7), tetrodoine (8), respectively. Conclusion Compounds 5, 7, 8 are isolated from Hippocampus trimaculatus Leach for the first time.