Background Leprosy, caused by Mycobacterium leprae, affects over 200,000 people annually worldwide and remains endemic in the ethnically diverse, mountainous and underdeveloped southwestern provinces of China. Delayed diagnosis of leprosy persists in China, thus, additional knowledge to support early diagnosis, especially early diagnosis of paucibacillary (PB) patients, based on the host immune responses induced by specific M. leprae antigens is needed. The current study aimed to investigate leprosy patients and controls in Southwest China by comparing supernatants after stimulation with specific M. leprae antigens in an overnight whole-blood assay (WBA) to determine whether host markers induced by specific M. leprae antigens improve the diagnosis or discrimination of PB patients with leprosy. Methodology/Principal findings Leprosy patients [13 multibacillary (MB) patients and 7 PB patients] and nonleprosy controls [21 healthy household contacts (HHCs), 20 endemic controls (ECs) and 19 tuberculosis (TB) patients] were enrolled in this study. The supernatant levels of ten host markers stimulated by specific M. leprae antigens were evaluated by overnight WBA and multiplex Luminex assays. The diagnostic value in PB patients and ECs and the discriminatory value between PB patients and HHCs or TB patients were evaluated by receiver operator characteristics (ROC) analysis. ML2044-stimulated CXCL8/IL-8 achieved the highest sensitivity of 100%, with a specificity of 73.68%, for PB diagnosis. Compared to single markers, a 3-marker combination model that included ML2044-induced CXCL8/IL-8, CCL4/MIP-1 beta, and IL-6 improved the diagnostic specificity to 94.7% for PB patients. ML2044-stimulated IL-4 and CXCL8/IL-8 achieved the highest sensitivity (85.71% and 100%) and the highest specificity (95.24% and 84.21%) for discriminating PB patients from HHCs and TB patients, respectively. Conclusions Our findings suggest that the host markers induced by specific M. leprae antigens in an overnight WBA increase diagnostic and discriminatory value in PB patients with leprosy, with a particularly strong association with interleukin 8. Author summary Leprosy, caused by Mycobacterium leprae, affects over 200,000 people annually worldwide and remains endemic in the ethnically diverse, mountainous and underdeveloped southwestern regions of China. Although it is curable, delayed diagnosis of leprosy persists in China, with a disability rate as high as 20% nationwide. To identify the source of infection and block transmission more effectively, further knowledge about the diagnostic value of antigen-specific induced host immune responses in patients is needed. The current study aimed to evaluate the diagnostic value of an overnight whole-blood assay for the diagnosis of paucibacillary (PB) leprosy patients, and differences in the ability of specific M. leprae antigens to stimulate a panel of host markers was tested by an overnight whole-blood assay. Our findings suggest that host markers induced by specific M. leprae antigens in an overnight WBA have diagnostic value in leprosy patients and discriminatory value between leprosy patients and healthy household contacts (HHCs) or tuberculosis (TB) patients.