Ergothioneine (EGT) is a high-value natural antioxidant that cannot be synthesized by the human body. This study showed that Rhodotorula mucilaginosa DL-X01 can use untreated molasses and fish bone meal enzymatic hydrolysate as the substrates to synthesize EGT. By optimizing the growth conditions, the EGT yield reached 29.39 mg/L when molasses and fish bone meal (FBM) were added at 60 g/L and 400 g/L respectively. Finally, the EGT yield was increased to 216.25 mg/L by fed-batch fermentation in a 5 L bioreactor. Compared with the fermentation by yeast extract peptone dextrose medium, the feedstock cost of EGT production was reduced by 330.91 % by using molasses and FBM as substrates. These results showed that R. mucilaginosa DL-X01 can produce high-value EGT using two cheap processing by-products, molasses and FBM, which is of great significance for environmental protection and sustainable development.