Efficient delivery of biomolecules into neurons has significant impacts on therapeutic applications in the central nervous system (CNS) and fundamental neuroscience research. Existing viral and non-viral delivery methods often suffer from inefficient intracellular access due to the endocytic pathway. Here, a neuron-targeting and direct cytosolic delivery platform is discovered by using a 15-amino-acid peptide, termed the N1 peptide, which enables neuron-specific targeting and cytosolic delivery of functional biomolecules. The N1 peptide initially binds hyaluronan in the extracellular matrix and subsequently passes the membrane of neurons without being trapped into endosome. This mechanism facilitates the efficient delivery of cell-impermeable and photo-stable fluorescent dye for super-resolution imaging of dendritic spines, and functional proteins, such as Cre recombinase, for site-specific genome modification. Importantly, the N1 peptide exhibits robust neuronal specificity across diverse species, including mice, rats, tree shrews, and zebra finches. Its targeting capability is further demonstrated through various administration routes, including intraparenchymal, intrathecal, and intravenous (i.v.) injections after blood-brain barrier (BBB) opening with focused ultrasound (FUS). These findings establish the N1 peptide as a versatile and functional platform with significant potential for bioimaging and advanced therapeutic applications.