Selenium (Se)-enriched Pleurotus ostreatus fungus powder with different total Se concentrations was used in combination with 5-FU to treat HepG2 hepatocellular carcinoma cells. The CCK-8 and flow cytometry double staining methods were applied to investigate the tumor suppressive effect of Se-enriched P. ostreatus fungus powder combined with 5-FU. The Se-enriched P. ostreatus fungus powder was then used as the raw material to develop a Se-enriched sugar substitute bread, providing scientific evidence for the development of Se-enriched functional foods. The results demonstrate that the Se-enriched P. ostreatus fungus powder had an inhibitory effect on the proliferation of HepG2 hepatocellular carcinoma cells, with the inhibition rate ranging from 13.89% to 27.39%. The number of apoptotic cells in HepG2 hepatocellular carcinoma increased in a concentration-dependent manner after treatment with different total Se concentrations of Se-rich P. ostreatus fungus powder combined with 5-FU for 36 h (p<0.05). The total apoptosis rates were 26.36%~49.72%, respectively, and the apoptosis rates were higher than those in the 5-FU group (p<0.05). The optimal processing formula of Se-enriched sugar-substitute bread was as follows: 0.06 g Se-enriched P. ostreatus fungus powder was added to 200 g flour with 25 g compound sugar substitute and baked at 175 °C for 20 min; the total Se content was 0.174±0.011 mg/kg. In conclusion, when HepG2 hepatoma cells were treated with Se-enriched P. ostreatus fungus powder at different total Se concentrations combined with 5-FU, HepG2 hepatoma cell growth was inhibited, increasing the efficiency of detoxification. The optimal processing formula of Se-enriched sugar substitute bread, which has various research and application values, was also developed.