Reducing the viscosity of molasses environmentally and selectively removing the harmful ingredients for microbes are the keys to promoting the bioavailability of molasses. A simple and environmental in situ pretreatment method integrating surfactants and alkali was developed to reduce the viscosity of molasses prior to l-lysine production using Escherichia coli ZY0217. Adding activated carbon and modified orange peel based on the in situ pretreatment process effectively removed pigments and excessive zinc in the molasses and also significantly increased the cell growth and l-lysine yield from E. coli ZY0217. The experimental results showed that a mixture of secondary alkane sulfonate, an anionic surfactant, and HodagCB-6, a non-ionic surfactant, effectively reduced the viscosity of the molasses more so than any single surfactant. When the surfactant mixture was added at a concentration of 0.04 g/L to the molasses, the omega value was 0.4, and when ammonia was added at 0.6 %, the lowest viscosity of 705 mPa center dot s was obtained. Further, 91.5 % of the color and 86.68 % of the original levels of zinc were removed using an activated carbon and modified orange peel treatment on the molasses with the lowest viscosity, which further promoted cell growth and l-lysine production. In the fed-batch cultivation process, the l-lysine concentration achieved using a constant-speed feeding strategy was 45.89 g/L, with an l-lysine yield of 27.18 %, whereas the l-lysine yield from untreated molasses was only 10.13 %. The increase in l-lysine yield was related to the reduced viscosity and the detoxification of the molasses. Lastly, the pretreatment was found to significantly enhance the conversion of sugars in the molasses to l-lysine.