Background: Some studies have shown that long-term exposure to job stress could result in burnout, and BDNF polymorphism may play an important role in its psychopathological mechanism. However, the inter-relationships between the job-related stress, serum BDNF level, BDNF genotype and job burnout have not been examined. This study was to explore the job stress x BDNF rs2049046 interaction and the role of serum BDNF level in job burnout in a Chinese Han population. Methods: Using a cross-sectional design, 205 healthy subjects were recruited from a public institution in Beijing and assessed for job stress using the House and Rizzo's Work Stress Scale, and job burnout using the Maslach Burnout Inventory (MBI). The BDNF rs2049046 polymorphism was genotyped and serum BDNF (sBDNF) levels were assayed in all of subjects. Results: The correlations between the job stress score and two burnout subscale scores (emotional exhaustion and cynicism) were significant (both p < 0.001), but not with professional efficacy. There were no significant main effects of the BDNF rs2049046 genotype on burnout, and no significant correlation was observed between sBDNF levels and job burnout. However, the interaction between the job stress and the BDNF rs2049046 genotype (F = 2.709, df = 2, 183, p = 0.032) or between the job stress and sBDNF levels on burnout was significant (t = - 2.132, p = 0.035). To be specific, the individuals with the BDNF rs2049046 AT genotype showed a greater susceptibility to the burnout cynicism compared to AA homozygote only in medium-stress group (F = 4.327, df = 1,117, p = 0.015). The individuals who had lower sBDNF showed higher burnout level than those who had higher sBDNF in low-stress group. Conclusions: Our findings suggest that the BDNF system may interact with job stress to affect burnout, showing that interaction between BDNF rs2049046 and job stress or the interaction between BDNF levels with work stress on certain burnout dimensions.