Cationic polymers have recently attracted considerable interest as research breakthroughs for various industrial and biomedical applications. They are particularly interesting due to their highly positive charges, acceptable physicochemical properties, and ability to undergo further modifications, making them attractive candidates for biomedical applications. Polyethyleneimines (PEIs), as the most extensively utilized polymers, are one of the valuable and prominent classes of polycations. Owing to their flexible polymeric chains, broad molecular weight (MW) distribution, and repetitive structural units, their customization for functional composites is more feasible. The specific beneficial attributes of PEIs could be introduced by purposeful functionalization or modification, long service life, biocompatibility, and distinct geometry. Therefore, PEIs have significant potential in biotechnology, medicine, and bioscience. In this review, we present the advances in PEI-based nanomaterials, their transfection efficiency, and their toxicity over the past few years. Furthermore, the potential and suitability of PEIs for various applications are highlighted and discussed in detail. This review aims to inspire readers to investigate innovative approaches for the design and development of next-generation PEI-based nanomaterials possessing cutting-edge functionalities and appealing characteristics.