Some short-chain fatty acids with a pungent or unpleasant odor are important components of human body odor. These malodors severely threaten human health. The antagonists of malodors would help to improve odor perception by affecting the interaction between odors and their receptors. However, the traditional odor detection and analysis methods, such as MOS, electrochemical, conductive polymer gas sensors, or chromatography-mass spectrometry are not suitable for screening the antagonists since they are unable to detect the ligand efficacy after odor-receptor binding. In this study, RT-PCR results showed that HL-1 cardiomyocytes endogenously express the olfactory receptor 558 (Olfr558) which can be activated by several malodorous short-chain fatty acids. Therefore, an in vitro HL-1 cardiomyocyte-based olfactory biosensor (HCBO-biosensor) was developed by combining cardiomyocytes and microelectrode array (MEA) chips for screening the potential antagonists of the Olfr558. Firstly, it showed that the biosensor specifically responded to ligands of Olfr558 through odor stimulation experiments. Then, an odor response model of HL-1 cardiomyocytes was constructed by a ligand of Olfr558 (isovaleric acid). The response feature of the in vitro HCBO-biosensor to individual odors and mixtures with a potential antagonist (citral or β-damascenone) were extracted and compared. Finally, the Olfr558-inhibited efficiency was indirectly detected by comparing the half-maximal inhibitory concentration of isovaleric acid. The results showed that β-damascenone greatly inhibited Olfr558 while citral showed no significant inhibitory effect. In conclusion, we built a novel screening method for the antagonists of Olfr558 based on HL-1 cardiomyocytes and the MEA chip which will assist odor-related companies to develop novel antagonists of Olfr558.