Novel symmetric biodegradable dendritic tri-block copolymer, consisting of a core of hydrophobic poly(L-lactide) (PLLA) block and 32 poly(L-lysine) (PLL) arms, was synthesized via a facile chemistry route. The as-synthesized tri-block copolymer was characterized with 1H NMR, water contact angle measurements, GPC test, and DSC analysis. The 1H NMR and GPC tests indicated that the tri-block copolymer, PLL-PLLA-PLL, with well-defined architecture was synthesized. The water contact angle measurements demonstrated that the incorporation of L-lysine improved prominently the hydrophilicity, and the DSC analysis indicated that the crystalline region of PLLA was slightly modified by the L-lysine component, decreasing melting temperature and crystallinity in the PLL-PLLA-PLL copolymer. NPCs and PC12 cells were seeded on PLLA, dendritic PLLA with 32 L-lysine terminals (d-PLLA-d), and PLL-PLLA-PLL films to investigate cell attachment, proliferation, and differentiation. The results strongly demonstrated that d-PLLA-d and PLL-PLLA-PLL films, especially PLL-PLLA-PLL films, dramatically promoted cell attachment, proliferation, and, more importantly, differentiation, particularly neurite outgrowth.