The invention discloses tobacco receptor protein genes as well as a cloning method and application thereof. The tobacco receptor protein genes include NtCOI1-1 and NtCOI1-2 with nucleotide sequences as shown in SEQ ID:NO.1 and SEQ ID:NO.2. The cloning method comprises the following steps of performing tobacco root cDNA synthesis: extracting tobacco root total RNA, and performing reverse transcription to obtain first chain cDNA; and performing PCR amplification of the genes NtCOI1-1 and NtCOI1-2: taking the tobacco root cDNA as a template, designing primers according to the sequences of the genes NtCOI1-1 and NtCOI1-2, performing PCR amplification, recovering and purifying PCR amplification products, and performing sequencing. The application is the application of the tobacco receptor protein genes NtCOI1-1 and NtCOI1-2 in a transgenic tobacco plant for regulating and controlling the nicotine content of tobacco. According to the invention, functional identification is carried out on thecloned genes NtCOI1-1 and NtCOI1-2 by using a CRISPR/CAS9 technology. Cloning identification of the tobacco genes NtCOI1-1 and NtCOI1-2 provides new target genes for regulating and controlling the nicotine content of the tobacco.