Real-time detection of bio-aerosol has become the focus of research in recent years. Based on the existing technology, the dual-channel bio-aerosol detection method which includes 280 nm and 365 nm channel is developed. With the dual-channel bio-aerosol detection system, the intrinsic fluorescence spectra and intensity of tryptophan and nicotinamide adenine dinucleotide (NADH) are detected respectively by deploying the ultraviolet source at 280 nm and 365 nm. On the basis of the result, the calibration method is determined, and then the analysis of bull serum albumin (BSA) and cigarette particles′ results are researched. The results demonstrate that it can improve the accuracy of particles pre-discrimination by using the dual-channel bio-aerosol detection technology. The technology has strong application value and development space.