This article reports a new approach to track (x, y, z, t) coordinates of multiple fluorescent particles (diameter range 1–10 μm) simultaneously using a quantitative defocusing method. We find that the defocused image of a 1-μm diameter fluorescent particle formed by the objective lens of a conventional microscope has a bright outer ring due to the spherical aberration of the lens system. The ring radius increases as the particle is moved away from its reference plane and closer to the lens. The reference plane refers to locations of the particle at which the projected image is in focus. The (x, y, z) coordinates of the particle are then inferred from the center location of the image ring as well as the ring radius. The described technique is implemented successfully for obtaining 3D trajectories of swimming Escherichia coli cells.