<p>It remains a great challenge to control weeds in rapeseed fields in China. Breeding herbicide-resistant rapeseed varieties and using corresponding herbicide formulations has become the most economical and effective way to control weeds in rapeseed field. Characterization of more herbicide-resistant genetic resources will provide opportunities for breeders to develop rapeseed herbicide-resistant varieties with good agronomic performance. Previously, we obtained the tribenuron methyl (TBM)-resistant mutant K4 from ZS9 (<i>Brassica napus</i> L.) through ethyl methyl sulfonate mutagenesis and TBM foliar-spray screening. In this study, the inheritance and molecular characterization of the mutant K4 are carried out. Genetic investigation indicated that the herbicide-resistance of the K4 was controlled by one dominant allele at a single nuclear gene locus. Molecular characterization showed that a single point substitution at position 535 from C to T in <i>BnAHAS3</i> (<i>BnAHAS3</i>^<i>535T</i>), which resulted in a mutation at point 179 in BnAHAS3. The K4 showed a certain degree of resistance to TBM, bensulfuron methyl, and monosulfon sodium, which were 50, 30, and 5 times that of ZS9, respectively. AHAS enzyme assay, structural analysis of AHAS proteins, affinity detection between TBM and BnAHAS3 by surface plasmon resonance analysis, and the transgenic experiment in Arabidopsis using <i>BnAHAS3</i>^<i>535T</i> confirmed that <i>BnAHAS3</i>^<i>535T</i> endow the K4 with herbicides resistance. In addition, an allele-specific marker was developed to quickly distinguish the heterozygous and homozygous mutated alleles <i>BnAHAS3</i>^<i>535T</i>. In conclusion, our research identified and characterized one novel mutative <i>AHAS</i> allele in <i>B. napus</i> and enriched genetic resource for developing herbicide-resistant rapeseed cultivars.</p>