The human eye is a sophisticated and sensitive sensory organ. Because of the existence of the blood-ocular barrier and corneal-scleral barrier, safe and efficient ocular drug delivery system is highly desired; yet, it remains an unsolved issue. Due to the unique structure and drug loading property, Poly(amidoamine) (PAMAM) has received much attention in the ocular drug delivery investigation. Herein, we evaluated the ocular cytotoxicity and biosafety of PAMAM dendrimers. The ocular cytotoxicity and biosafety of PAMAM dendrimers were evaluated by conducting and experiments on ocular systems. The effect of PAMAM dendrimer of different generations (G4.0, G5.0, and G6.0) and concentrations on ocular cell metabolism, apoptosis, and oxidative damage were quantitatively assessed. biosafety of PAMAM dendrimers were further investigated on intraocular tissue by ocular irritation and intravitreal injection approaches. It is found that that the cytotoxicity of PAMAM was time and generation dependent. PAMAM at a concentration below 50 μg/mL had minimal impact on the ocular tissue, whereas it caused apparent damage when above 50 μg/mL in the investigated situation. Further, our results showed that higher concentration of dendrimer (100 μg/mL) was associated with functional impairment demonstrated via optical coherence tomography and electroretinogram, although macroscopic structural changes were absent in fundus and histopathological studies. Overall, a higher concentration of PAMAM, such as above 50 μg/mL, may cause ocular functional damage. The PAMAM at the concentrations lower than 50 μg/mL showed good biocompatibility and biosafety in human ocular cells and tissues.