Nanoparticles are rapidly emerging as therapeutic delivery vectors defined by size-dependent properties. They offer several advantages over the traditional drug-delivery systems and medical diagnostics but also pose considerable challenges for systemic applications. Gene delivery is one of the important applications of nanotechnology. Usually, the nanoparticles employed for gene delivery are either formed by condensation of DNA with preformed cationic polymers or by polymerization of monomeric units thereby entrapping DNA in it. The physicochemical properties such as size, shape, surface morphology have been found to have significant influence on the gene-delivery efficacy of nanoparticles. Furthermore, when administered in vitro and in vivo, the efficiency of nanoparticles depends on a wide variety of other parameters, that is, transfection conditions, time of exposure, cell type and so forth. In this review, the potential role of characterization of nanoparticles physicochemical properties on the in vitro and in vivo gene delivery efficacy of nanoparticles is discussed.