A large number of different polymers have been developed and studied for application as DNA carriers for non-viral gene delivery, but the DNA binding properties are not understood. This study describes the efficiency of nanoparticle formation by time-resolved fluorescence measurements for poly(β-amino esters), cationic biodegradable polymers with DNA complexation and transfection capability. From the large library of poly(β-amino esters) ten polymers with different transfection efficacies were chosen for this study. The binding constants for nanoparticle formation were determined and compared to polyethylene imines with the same method. Although the DNA binding efficiency of the amine groups are similar for both types of polymers, the overall binding constants are an order of magnitude smaller for poly(β-amino esters) than for 25 kDa polyethylenimines, but yet poly(β-amino esters) show comparable DNA transfection efficacy with polyethyleneimines. Within this series of polymers the transfection efficacy showed increasing trend in association with relative efficiency of nanoparticle formation.