Poly(ethylene glycol)-poly(alanine-co-phenylalanine) (PEG-PAF) block copolymers with similar molecular weights were synthesized to investigate the effect of the partial incorporation of d-alanine into PEG-l-PAF. The ratio of l-alanine to d-alanine of the polymer varied over 100/0, 80/20, 60/40, 50/50, and 0/100. Circular dichroism and FTIR spectra indicated that the PEG-PAFs with the mixed composition of l-alanine and d-alanine exhibited dominantly random coil structures, whereas the PEG-PAF with the enantiomeric alanine (100/0 or 0/100) exhibited right-handed or left-handed α-helical structures as well as β-sheet structures, respectively. Dynamic light scattering of the polymer aqueous solution indicated that the size of nanoassemblies significantly decreased as a result of the partial incorporation of d-alanine into PEG-l-PAF. The most probable diameters were 20-40 nm and 80-105 nm for the PEG-PAFs with a mixed composition (l-alanine/d-alanine ratios of 80/20, 60/40, and 50/50) and an enantiomeric composition (l-alanine/d-alanine ratios of 0/100 and 100/0), respectively. As the temperature increased, the relative β-sheet content decreased for PEG-PAF with the enantiomeric alanine, while there was no significant change in random coil structures of PEG-PAFs with a mixed composition. 13C-NMR spectra suggest the dehydration and decrease in molecular motion of PEG during the sol-to-gel transition of PEG-PAF. Through the partial incorporation of d-alanine into PEG-l-PAF, thermogelling behaviour was also observed at much higher temperatures and concentrations due to the difference in the secondary structure and nanoassembly of the polymers. The enzymatic degradability of PEG-PAF also decreased as a result of the partial incorporation of d-alanine into PEG-l-PAF. The paper suggests that partial incorporation of d-alanine into an l-polypeptide block copolymer can be a useful method in designing a biodegradable material as well as controlling nanoassemblies of the polymer.