•A self-driving bead-based bioassay.•High temporal stability and reproducibility (<1.2%).•Free from background fluctuations with the use of reference particles.•Dose-dependent measurement of microorganisms, as low as 106 cfu/mL.•Detection of live microorganisms, including motile bacterium, P. aeruginosa, and nonmotile bacterium, S. aureus. A self-driving, thermal-diffusion-based bioassay for the detection of microorganisms in a liquid medium is presented in this paper. In the bioassay, each particle functions as an individual sensing probe. Thus, the representative Brownian velocity of microparticles can be obtained by analyzing the velocity histogram of each particle. The ensemble average was used to enhance peak Brownian velocity. Relative error was reduced to 0.5% when the number of counted particles exceeded 60. The experimentally measured and theoretically derived Brownian velocities and diameters of the particles were in good agreement. The relative standard deviations of the temporal stability and reproducibility of the bioassay were maintained below 1.2%. A calibration curve was constructed and used to distinguish two mixed colloidal suspensions to provide proof that the bioassay can be used in practical applications. The particles were functionalized with antibodies to enable the real biological application of the bioassay in the capture and detection of motile Pseudomonas aeruginosa and nonmotile Staphylococcus aureus. The diffusivity values of both bacterial growth media decreased as bacterial concentration increased. Given that the viscosity of the growth media varied as bacteria proliferated, additional bacteria-free reference particles were added to the medium to provide dynamic background information. The diffusion-based bioassay presented here is easy to use, robust, and highly reliable. In contrast to most existing biosensors, it does not require an external power source and is thus ideal for use in resource-limited areas.