The production of microbial lipid using lignocellulosic agroforestry residues has attracted much attention. But, various inhibitors such as phenols and furans, which are produced during lignocellulosic hydrolysate preparation, are harmful to microbial lipid accumulation. Herein, we developed a novel detoxification strategy of rice straw hydrolysate using immobilized laccase on magnetic Fe3O4 nanoparticles for improving lipid production of Rhodotorula glutinis. Compared with free laccase, the immobilized laccase on magnetic nanoparticles showed better stability, which still retained 76% of original activity at 70 degrees C and 56% at pH 2 for 6 h. This immobilized laccase was reused to remove inhibitors in acid-pretreated rice straw hydrolysate through recycling with external magnetic field. The results showed that most of phenols, parts of furans, and formic acids could be removed by immobilized laccase after the first batch. Notably, the immobilized laccase exhibited good reusability in repeated batch detoxification. 78.2% phenols, 43.8% furfural, 30.4% HMF, and 16.5% formic acid in the hydrolysate were removed after the fourth batch. Furthermore, these detoxified rice straw hydrolysates, as substrates, were applied to the lipid production of Rhodotorula glutinis. The lipid yield in detoxified hydrolysate was significantly higher than that in undetoxified hydrolysate. These findings suggest that the immobilized laccase on magnetic nanoparticles has a potential to detoxify lignocellusic hydrolysate for improving microbial lipid production.