This contribution describes a simple, aerosol‐based method for fabricating monodisperse particles containing mixtures of poly(lactide‐co‐glycolic acid) [PLGA], protamine sulfate (Prot), and poly(l‐lysine) [PLL] as nanocarriers for gene transfection. Aqueous solutions of PLGA, Prot, and PLL were collison‐atomized, and the resulting aerosolized droplets were dried “on the fly” to form solid particles, which then were electrostatically size‐classified into 50, 100, and 200 nm mobility diameter samples. Measurements of cell viability and transfection reveal that the fabricated nanocarriers have a lower cytotoxicity (>85% in cell viability) and a higher transfection efficiency [>8.7 × 105 in relative light units (RLU) mg−1] than does 25 kDa polyethyleneimine (≈50% and 6.8 × 105 RLU mg−1).