Cross-linked layer-by-layer (LbL) assemblies with precisely tuned surface zeta potential were fabricated to control the adsorption of proteins, cells and bacteria for different biomedical applications. Two weak polyions including a synthetic polyanion and polyethylenimine (PEI) were assembled under controlled conditions and cross-linked to prepare three robust LbL films as model surfaces with similar roughness and water affinity but displaying negative, zero and positive net charges at the physiological pH (7.4). These surfaces were tested for their abilities to adsorb proteins including Bovine Serum Albumin (BSA) and Lysozyme (LYZ). In adsorption tests, the LbL films bind more proteins with opposite charges but less with like charges, indicating that electrostatic interactions play a major role in protein adsorption. However, LYZ showed higher non-specific adsorption than BSA because of the specific behavior of LYZ molecules such as stacked multilayer formation during adsorption. To exclude such stacking effects from experimens when assessing adsorption by AFM, protein molecules were covalently immobilized on AFM colloidal probes to measure the adhesion forces against the model surfaces utilizing direct protein molecule-surface contacts. The results confirmed the dominating role of electrostatic forces in protein adhesion. In fibroblast cell and bacteria adhesion tests, similar trends (high adhesion on positively charged surfaces, but much lower on neutral and negatively charged surfaces) were observed because the fibroblast cell and bacterial surfaces studied possess negative potentials. The cross-linked LbL films with improved stability and engineered surface charge described in this study provide an excellent platform to control the behavior of different charged objects and can be utilized in practical biomedical applications.