Laccase is a green biocatalyst. It works with molecular oxygen and produces water as the only by-product. However, its practical application is far less than satisfactory due to the low stability/poor reusability of free laccase and the potential secondary pollution caused by dissolved mediators. To address those bottlenecks in laccase-based catalysis, a novel biocatalyst (Immo-LMS) was fabricated by simultaneously immobilizing both laccase and a mediator (acetylacetone, abbreviated as AA) into a hydrogel through the laccase-AA initiated polymerization. This self-initiated immobilization process avoided the forced conformational change of laccase in the passive embedding to pre-existing carriers. Resulting from the effective cooperation of laccase and AA, the Immo-LMS had the highest substrate conversion quantity to malachite green, followed by the sole immobilized laccase and the immobilized lac case with an external mediator. Besides the improved activity, the Immo-LMS showed enhanced stability. The good performance of the Immo-LMS suggests that the co-immobilization of laccase and mediator through the self-initiated one-pot process was a promising strategy for the immobilization of laccase, which is expected to be helpful to cut down the running cost as well as the potential toxicity that come from mediators in the practical application of laccase. (C) 2016 Elsevier Inc. All rights reserved.