This paper presents a modeling and experimental framework to characterize the chemotaxis of Serratia marcescens (S. marcescens) relying on two-dimensional and three-dimensional tracking of individual bacteria. Previous studies mainly characterized bacterial chemotaxis based on population density analysis. Instead, this study focuses on single-cell tracking and measuring the chemotactic drift velocity V(C) from the biased tumble rate of individual bacteria on exposure to a concentration gradient of l-aspartate. The chemotactic response of S. marcescens is quantified over a range of concentration gradients (10^{-3} to 5 mM/mm) and average concentrations (0.5 × 10(-3) to 2.5 mM). Through the analysis of a large number of bacterial swimming trajectories, the tumble rate is found to have a significant bias with respect to the swimming direction. We also verify the relative gradient sensing mechanism in the chemotaxis of S. marcescens by measuring the change of V(C) with the average concentration and the gradient. The applied full pathway model with fitted parameters matches the experimental data. Finally, we show that our measurements based on individual bacteria lead to the determination of the motility coefficient μ (7.25 × 10(-6) cm(2)/s) of a population. The experimental characterization and simulation results for the chemotaxis of this bacterial species contribute towards using S. marcescens in chemically controlled biohybrid systems.