Ligninolytic fungi and enzymes (i.e., laccase, manganese peroxidase, and lignin peroxidase) have been applied recently in the production of second-generation biofuels. This review contains the analysis of ligninolytic enzymes and their applications in second-generation biofuels. In here, each of the ligninolytic enzymes was described analyzing their structures, catalysis, and reaction mechanism. Additionally, delignification and detoxification, the two most important applications of ligninolytic enzymes, were reviewed and analyzed. The analysis includes an evaluation of the biochemical process, feedstocks, and the ethanol production. This review describes the current situation of the ligninolytic enzymes technology and its future applications in bioethanol industry.