Biopolymer microparticles have been developed for applications that require biocompatibility and biodegradability, such as drug delivery. In this study, we assessed the production of microparticles using carnauba wax, κ-carrageenan, alginate, and poly (lactic-co-glycolic acid) (PLGA) with the aim of developing a novel, DNA-tracer-loaded, biopolymer surrogate with a size, shape, surface charge, and relative hydrophobicity similar to stationary-phase to mimic the bacteria's mobility and persistence in engineered water systems. We found that the type and concentration of biopolymer, reaction conditions, and synthesis methods affected the morphology, surface charge, relative hydrophobicity, and DNA tracer loading efficiency of the biopolymer microparticles produced. Carnauba wax, κ-carrageenan, and alginate (Protanal, and low and medium viscosity) produced highly polydisperse microspheres. In contrast, PLGA and alginate-CaCO produced uniform microspheres and rod-shaped microparticles, respectively, with high DNA tracer loading efficiencies (PLGA 70% and alginate-CaCO 95.2 ± 5.7%) and high reproducibilities. Their synthesis reproducibility was relatively high. The relative hydrophobicity of PLGA microspheres closely matched the cell surface hydrophobicity of but not the bacterial morphology, whereas the polyelectrolyte layer-by-layer assembly was required to enhance the relative hydrophobicity of alginate-CaCO microparticles. Following this surface modification, alginate-CaCO microparticles represented the best match to in size, morphology, surface charge, and relative hydrophobicity. This new biopolymer surrogate has the potential to be used as a mimic to study the mobility and persistence of in water systems where the use of the pathogen is impractical and unsafe.