Recombinant human endostatin (MES), showing potent inhibition on angiogenesis and tumour growth, has great potential as a therapeutic agent for tumours. The aim of this study was to evaluate the biophysical and biological characterization of PEGylated recombinant human endostatin (M ES). Recombinant human endostatin was mono-PEGylated by conjugation with methoxy polyethylene glycol aldehyde (mPEG-ALD), and the modification site was identified by digested peptide mapping and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The purity was assessed by SDS-PAGE, high-performance liquid chromatography (HPLC), and capillary zone electrophoresis. The physicochemical property was analyzed through fluorescence spectroscopy, and circular dichroism. The bioactivity and anti-tumour efficacy of M ES were evaluated using an in vitro endothelial cell migration model and a null-mouse xenograft model of a prostatic cancer, respectively. M ES molecules contain a single 20 kDa mPEG-ALD molecule conjugated at the N-terminal portion of MES. The purity of M ES was greater than 98%. The physicochemical analysis demonstrated that PEGylation does not change the secondary and tertiary structure of MES. Notably, M ES retards endothelial cell migration and tumour growth when compared to control group. These biophysical and biological characterization study data contribute to the initiation of the ongoing clinical study.