Polymicrobial biofilms are major complications of various chronic infections. Therefore, in vitro biorelevant polymicrobial biofilm models are essential tools for medical studies. This study presents an in vitro model for dual species biofilm of Pseudomonas aeruginosa and Staphylococcus aureus developed on cell-derived matrices (CDMs), in order to simulate the microenvironment of in vivo biofilms. P. aeruginosa and S. aureus are two of the most frequent pathogens in polymicrobial biofilms of wound infections. Although they are commonly isolated from polymicrobial biofilms, their interaction is antagonistic; and there is severe battle between them for nutrients and space. We introduced a nutritious formulation supporting co-cultures of P. aeruginosa and S. aureus in order to study the interaction of these gram-positive and gram-negative bacterial species. Quantitative analyses demonstrated that the enrichment of tryptic soy broth (TSB) with NaCl and glucose facilitate dual-species biofilm formation of P. aeruginosa and S. aureus when it is mixed with fetal bovine serum (FBS). Furthermore, the dual species biofilm was incubated on CDMs. Characterization of the model by fluorescent and electron microscopy techniques revealed realistic features of chronic multi-species biofilms, including competitive distribution pattern of two bacterial species and small-colony variants (SCVs) morphology of S. aureus.