Objective To explore Mycobacterium tuberculosis（MTB） infection and the expression of the Notch signaling pathway in patients with primary lung cancer. Methods A total of 96 patients with primary lung cancer who underwent radical surgery at Chongqing University Cancer Hospital between January 2022 and June 2022 were enrolled as research subjects. MTB and its L-forms in the lung tissue were detected using in situ molecular hybridization and acid-fast staining. Drug resistance was analyzed using a drug susceptibility test. Mutations at the sites of target genes were identified using sequencing. The expressions of Notch1，Notch2 and Notch3 proteins in the lung tissue were determined using Western blot. Results Of the 96 patients with primary lung cancer，the positive rate of MTB-L forms was 38.54%（37/96），with 68 MTB-L strains isolated from the sputum samples of the positive patients. The resistance rate of the isolated MTB-L strains to first-line anti-tuberculosis drugs was 60.29%（41/68），with the highest drug resistance rate of 22.06% for the combination of isoniazid，streptomycin，rifampicin，and ethambutol. The resistance rate of the isolated MTB-L strains to second-line anti-tuberculosis drugs was 36.76%（25/68），with the highest drug resistance rate of 14.70% for ofloxacin alone. The target genes with the top three mutation rates at the drug sites of the MTB-L strains were isoniazid katG S315T（45.58%），streptomycin rpsL K43R（42.65%），and rifampicin rpoB S450L（20.59%）. The relative expression levels of Notch1，Notch2，and Notch3 proteins in the lung tissue of patients with positive MTB-L infections were higher than those in the lung tissue of patients with negative MTB-L infections（P<0.05）. Conclusion MTB infection in patients with primary lung cancer is mainly caused by MTB L-forms，and drug resistance is severe. Abnormal activation of the Notch signaling pathway is observed in patients with MTB-L infection.