Benzo[a]pyrene (B[a]P) is a main toxic chemical component with tobacco smoking, which is a carcinogen to humans according to the adequate evidence from animals. 3-hydroxybenzo[a]pyrene (3-OHB[a]P) is a biomarker of B[a]P exposure. 3-OHB[a]P could be used to distinguish the populations with different levels of exposure, such as smokers, nonsmokers, occupational exposure population, and general population, and to determine the differences in metabolism. The research progress in 3-OHB[a]P is reviewed. Firstly, the carcinogenicity of B[a]P is discussed. And then the source of 3-OHB[a]P as well as the determination method of 3-OHB[a]P are reviewed. The applications of 3-OHB[a]P in biological monitoring are elaborated in detail, including occupational exposure population, smoking population, and the general population. Starting from the exist problems and current research status on 3-OHB[a]P, the prospects for applying 3-OHB[a]P to the study of biological monitoring smokers and general population are analyzed.