Optical tweezers have emerged as a powerful tool with broad applications in biology and physics. In force-measuring applications, the trapped bead position is usually accurately determined by forward-scattered detection. The current study discusses both backward-scattered detection and forward-scattered detection related to the linear detection range for a 3 μm bead and the distance between the two laser system focuses, confirming the optimum positions of the two focuses. The result indicates that the linear detection range of backward-scattered detection is longer than the forward-scattered one. Finally, this work investigates real-time adhesion force measurements between human macrophages and 3 μm trapped beads coated with lipopolysaccharides at various pH values by optical tweezers with backward-scattered detection.