Background: Mactra veneriformis, a typical marine bivalve mollusk, delicious sea food while low cost, is ubiquitous and abundant in Chinese coastal areas, especially in the coastal shoals of Jiangsu province. To our knowledge, previously reported analytical methods can not meet a set of quality control. Objective: For the simultaneous determination of eight components (uridine, inosine, guanosine, thymidine, adenosine, xanthine, thymine and hypoxanthine) in M. veneriformis, a high performance liquid chromatography with UV detector method was established. Materials and Methods: To develop the method, a reverse phase column, BioBasic-C 18 (5 μm, 4.6 mm × 250 mm) was used. The mobile phase consisted of methanol and water using a gradient elution. The UV wavelength was set at 245 nm. The analysis conditions including extraction methods, extraction solvents, and HPLC parameters were optimized systematically for achieving good separation. Linearity, accuracy, repeatability and detection limit was revealed and showed good performance. Results: The optimized HPLC method was successfully applied for the qualititation of 5 nucleosides namely, uridine, inosine, guanosine, thymidine, adenosine and 3 nucleobases namely, xanthine, thymine, hypoxanthine in M. veneriformis. Conclusion: A method with less time-consuming, more sensitive, and more precise was developed for the quantitative determination of nucleosides and nucleobases in M. veneriformis extractions. The established method might apply as an alternative approach for the quality assessment of M. veneriformis.