Time-averaged flow velocity and pulsating flow velocity coexist in actual turbulence. In order to study the effect of turbulent pulsating flow velocity on the physical and chemical properties of biofilm, an oscillating-grid biofilm reactor was designed which only has pulsating flow velocity with the time-averaged flow velocity being zero. The two-dimensional turbulent flow field of the reactor was measured by particle image velocimetry (PIV) at five oscillating-grid frequencies, and the effective turbulent intensities at biofilm carriers were calculated. The biomass, thickness, and density of the biofilm, as well as the composition of the extracellular polymeric substances (EPS) in the oscillating-grid reactor were measured and analyzed at different turbulent pulsating flow velocities. Research results show that changing the turbulent pulsating flow velocity would change the physical and chemical properties of biofilm. With the increase of the turbulent pulsating velocity, the biomass and thickness of the biofilm first increased and then decreased, and the maximum value was reached when the effective turbulent intensity was 1.92 cm/s. While the biofilm density and the content of protein and carbohydrates in EPS increased with increasing turbulent pulsating flow velocity, and the content of protein and carbohydrates in tightly bound EPS (TB-EPS) was more than that in loosely bound EPS (LB-EPS). The ratios of protein to carbohydrates content in LB-EPS, TB-EPS, and total-EPS were greater than one. This research can provide theoretical reference for the design and operation optimization of biofilm reactors.