The black queen cell virus (BQCV) is a common agent that causes covert infection in hives and has a global distribution. In this study, TaqMan probe and EvaGreen (EG) dye-based real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays based on the amplification of coding senquences for the BQCV structural protein (SP) and the non-structural protein (NSP) were developed and validated for the detection of BQCV in adult bee and larvae/pupae. Additionally, the performances of commonly used EG and TaqMan chemicals for real-time qRT-PCR analyses were compared. The operating range for BQCV-SP TaqMan and EG real-time qRT-PCRs ranged 2.61–7.61 log10 RNA copies/reaction and 2.61–7.79 log10 RNA copies/reaction, respectively. The operating range for BQCV-NSP TaqMan and EG real-time qRT-PCRs ranged 6.83–11.83 log10 RNA copies/reaction and 6.98–11.98 log10 RNA copies/reaction, respectively. Based on these novel assays, the prevalence of BQCV in adult bees and larvae/pupae was 84% (88/105) and 44% (46/105), respectively. BQCV viral load was also within the operating range determined during assay validation. Comparable results were obtained in analytical and diagnostic performance analyses of the EG dye and TaqMan probe chemicals. Highly sensitive and analytically specific for the detection of BQCV, these real-time qRT-PCR assays will benefit the etiological and epidemiological studies of BQCV.