Despite progress in understanding diazotrophic distribution in surface soils, few studies have investigated the distribution of diazotrophic bacteria in deeper soil layers. Here, we leveraged high-throughput sequencing (HTS) of nifH genes obtained to assess the influence of biochar amended soil (BC) and control (CK), and soil depths (0-20, 20-40 and 40-60 cm) on diazotrophic abundance and community structures, soil enzyme activities and physio-chemical properties. Multivariate ANOVA analysis revealed that soil depth had profound impact on majority of the soil parameters measured than fertilization. Although soil physio-chemical properties, enzymes activities, diazotrophic genera and enriched operational taxonomic units (OTUs) were significantly influenced across the entire soil profiles, we also observed that BC amended soil significantly increased cane stalk height and weight, nitrate (NO3-), ammonium (NH4+), organic matter (OM), total carbon (TC) and available potassium (AK), and enhanced diazotrophic genera in soil depth 0-20 cm compared to CK treatment. Soil TC, total nitrogen (TN), OM and NH4+ were the major impact factors shifting diazotrophic community structures in soil depth 0-20 cm. Overall, these results were more pronounced in 0-20 cm soil depth in BC than CK treatment.