Antibody incorporation in polydiacetylene (PDA) liposome was used to specifically detect Salmonella. Modifying physical properties of PDA liposome, such as size, surface charge, and packing of lipids, could affect the sensitivity and the stability of it. Therefore, the main goal of this study was to evaluate four factors simultaneously in PDA/antibody vesicles to detect Salmonella. Raman spectra were carried out to confirm covalent binding between antibody and PDA. The work followed through concomitant study of incubation temperature, incubation time, pH and antibody concentration allowing the development of some models that explain those variables related to colorimetric response, size and zeta potential. The smallest particle size (208 nm) showed the highest colorimetric response (90%), indicating PDA size enhanced sensitivity. Besides that, there is a relation between size and zeta potential: while particle size improved (from 300 nm to 700 nm), zeta potential enlarged too (from -30 to -20). This effect could be explained by PDA aggregation over time, by high temperature effect or by Salmonella presence. The study of zeta potential helped to understand that the interaction between Salmonella and antibody was through affinity between them. Therefore, understanding the parameters is important to improve PDA biosensors to apply in food industry and laboratory areas.