Abstract: Selenium is an essential micronutrient for humans with a narrow safe intake range. Inorganic selenium can be converted into readily absorbable organic selenium by Lentinula edodes, yet systematic comparisons of selenium enrichment patterns and dietary risks under multiple selenium sources and flushes are lacking in existing studies. To screen safe and efficient selenium sources for cultivation, Lentinula edodes strain 9608 was used as test material. Sodium selenite, sodium selenate and selenium-enriched yeast were supplemented into cultivation substrates at five mass concentration gradients of 0.50~10.00 mg·kg-1. Total selenium and selenoamino acids in caps and stipes harvested from two flushes were determined. An in vitro gastrointestinal digestion method was adopted to measure selenium bioaccessibility, and dietary safety was evaluated combined with estimated daily intake and target hazard quotient. The results were shown as follows: Selenium contents of shiitake mushrooms were significantly increased by all three selenium sources. Caps exhibited stronger selenium accumulation capacity than stipes, and higher selenium concentrations were detected in the first flush compared with the second flush. Under identical substrate selenium mass concentrations, the selenium accumulation capacity was ranked as sodium selenite > sodium selenate > selenium-enriched yeast. The proportion of selenoamino acids reached 78.52~97.63 wt.% in the second flush, indicating more sufficient biotransformation of organic selenium. Higher selenium bioaccessibility was observed in the gastrointestinal digestion phase than in the gastric phase, and better overall performance was obtained in the second flush. Under the selenium mass concentration gradients designed in this experiment, no dietary risks were observed when substrate selenium supplementation was ≤1.00 mg·kg-1, whereas potential exposure hazards existed at concentrations ≥2.00 mg·kg-1. Considering enrichment efficiency, organic selenium conversion rate and edible safety, the supplementation of sodium selenite at 0.50~1.00 mg·kg-1 in substrates is recommended for standardized selenium-enriched shiitake cultivation. High-selenium fruiting bodies can be applied to extract selenium functional active substances. Data support is provided by this study for the production and safety regulation of selenium-enriched shiitake mushrooms.