Injectable hydrogels have attracted great research interest for the delivery drugs and recruitment of cells to promote tissue regeneration. In this work, a series of the amino acid based methoxy-poly (ethylene glycol)-b-(poly (γ-ethyl-l-glutamate-co/b-l-phenylalanine) block copolymers (mPEG-b-P (ELG-co/b-Phe)s) with similar block chain length, but different proportion and sequence of γ-ethyl-l-glutamate (ELG) and l-phenylalanine (Phe) arrangements were prepared through ring-opening polymerization method. The influence of the ratio and sequence structures of mPEG-b-P (ELG-co/b-Phe)s on their thermosensitive aqueous gelation behaviors were investigated in detail. The results showed that the obtained polymers showned higher modulus (G’) and lower critical gelation temperature with higher proportion of l-phenylalanine. However, the physical properties start to deteriorate as the copolymers have a large number of phenylalanine groups. To clarify this phenomenon, the analysis of secondary structure with circular dichroism (CD) and fourier transform infrared (FTIR) spectrum showed that random coil reached a higher percentage as incorporated phenylalanine groups increased. On the other hand, the polymers with different sequence and similar compositions exhibited divergence due to the influence of random coil conformation. In addition, the good biocompatibility of mPEG-b-P (ELG-co-Phe)s were observed in vitro and in vivo. The copolymers have the potential to be used as thermo-sensitive injectable hydrogels for biomedical applications.