Nerve Growth Factor (NGF) is one of the members of the neurotrophin family with multifaceted functions. However, clinical application of NGF is hurdled by the challenge on formulation development. The objective of this study was to develop novel high-density lipoproteins (HDL)-mimicking nanoparticles (NPs) coated with α-tocopherol to incorporate NGF by a self-assembly approach. The NPs were prepared by an optimized self-assembly method that is simple and scalable. The composition of HDL-mimicking NPs was optimized. The prototype of the HDL-mimicking α-tocopherol-coated NPs contained phosphatidylserine (a negative charged phospholipid) and d-α-Tocopheryl polyethylene glycol succinate (a source of vitamin E) to enhance the entrapment efficiency of apolipoprotein A-I in the NPs. The entrapment efficiency of apolipoprotein A-I was about 30%. The NPs had particle size about 200 nm with a relatively narrow size distribution. Finally, cationic ion-pair agents were optimized to form ion-pairs with NGF to facilitate the incorporation of NGF into the NPs. Protamine sodium salt USP formed an optimal ion-pair complex with NGF. The results showed that the novel HDL-mimicking α-tocopherol-coated NPs successfully encapsulated NGF with over 65% entrapment efficiency by using this ion-pair strategy. In vitro release studies demonstrated a slow release of NGF from NGF NPs in PBS containing 5% BSA at 37 °C for 72 h. Further biodistribution studies showed that intravenously injected NGF NPs significantly increased NGF concentration in plasma and decreased the uptake in liver, spleen and kidney, compared to free NGF in mice.