Despite the advantages of stem cell injection to the skin wound site, this technique is limited in terms of the delivery of therapeutically functional stem cells to the wound site. Considering the abnormal microenvironment of a wound site, even a small damage or loss of cell function induced by severe trypsin treatment might lead to decrement of the therapeutic efficacy of stem cells. In this study, we have synthesized pH-responsive polymer mixed poly(amino ester) (mPAE), which can provide the appropriate cell culture condition for cell growth without cytotoxicity along with the maintenance of extracellular matrix after detachment using low pH (6.0) PBS. Human adipose derive stem cells (hADSCs) cultured on and detached from mPAE-coated tissue culture plates (TCPs) showed similar cell adhesion and angiogenic paracrine factor secretion, compared to hADSCs cultured on and detached from TCPs with trypsin. As a result, hADSCs detached with pH 6.0 PBS from mPAE-coated TCPs showed similar therapeutic angiogenesis and wound regeneration, compared to the hADSCs detached with trypsin from TCPs. In conclusion, our pH-responsive polymer, mPAE, might be utilized as an enzyme-free cell detachment system for future tissue engineering.