Targeted therapy for the central nervous system (CNS) has traditionally relied on intravenous injections or direct intracerebroventricular delivery. Recently, neuronal transport-mediated delivery from the periphery to the CNS, such as intranasal, has emerged as a promising alternative. The rabies virus is capable of efficiently entering the CNS via the neuromuscular junction, intra-axonal retrograde transport and synaptic cleft crossing, thus bypassing the restrictive blood-brain barrier (BBB). RVG29, a peptide derived from the rabies virus glycoprotein, exhibits excellent neurotropic properties. Inspired by this concept, we designed a novel brain-targeted system, PAA-PEG-RVG29, consisting of poly(amido amine)s (PAAs) functionalized with poly(ethylene glycol) (PEG) to reduce cytotoxicity and aggregation, and conjugated with RVG29 to enhance neuronal transport capability. Moreover, Rhodamine B (RhB) was loaded into this system as a model cargo by intermolecular hydrogen bonding, and its efficiency for intra- and inter-neuronal distribution was evaluated. PAA-PEG-RVG29 (RhB) exhibited a hydrodynamic radius of 29.9 nm, a zeta potential of +25 mV, and a RhB loading capacity of 36.4 μg/mg. Cytotoxicity and cellular uptake studies demonstrated promising biocompatibility and efficient internalization in Neuro2a, NSC34, and primary neurons. Furthermore, patch-clamp electrophysiology confirmed that there was no significant alteration effect on primary spinal neuronal action potential generation and propagation. In a two-compartment microfluidic chamber, PAA-PEG-RVG29 (RhB) exhibited effective retrograde axonal transport. A three-compartment chamber further showed progressive trans-synaptic delivery to neighboring neuronal somas. These results highlight the potential of PAA-PEG-RVG29 (RhB) for neuronal transport and synaptic cleft crossing, offering a promising strategy for neuronal delivery, bioimaging, and tracking.