The present invention discloses thermophilic ethanol-resistant β-glucosidase and an encoding gene and application thereof. The amino acid sequence of the thermophilic ethanol-resistant β-glucosidase of the present invention is as shown in SEQ ID NO: 2, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO: 1. The nature of the thermophilic ethanol-resistant β-glucosidase of the present invention is conductive to the application of cellulose high-temperature simultaneous saccharification and fermentation, the enzyme has a relatively high enzyme activity and obvious active effect, cellobiose is kept at low concentration level during the whole fermentation period, the inhibition of terminal products can be effectively eliminated, the thermophilic ethanol-resistant β-glucosidase can be applied on the process of fuel ethanol production, and the important application value of the thermophilic ethanol-resistant β-glucosidase of the present invention in the aspect of bioenergy is further indicated.