To investigate the pharmacokinetics of PEGylated recombinant human endostatin (M2ES) in rhesus monkey. M2ES was administered to rhesus monkeys by intravenous bolus injection, and serum M2ES concentration was determined by a self-developed ELISA assay. Pharmacokinetic parameters were calculated using a non-compartmental model of WinNonlin V2.1A software. The standard curve of self-developed ELISA assay was fitted by four-parameter method. The limit of detection (LOD) and LOQ were 0.3050 ng/mL and 0.9140 ng/mL, respectively. Following IV infusions of M2ES at 0.3, 1, and 3 mg/kg in rhesus monkeys, the serum M2ES concentration-time curve was fitted with a non-compartment model. The pharmacokinetic parameters were evaluated as follows: Terminal elimination half-life (T1/2) of M2ES were 3.30 ± 0.70, 29.50 ± 18.80 and 24.60 ± 8.90 h. Systemic clearance (CLsys) of M2ES were 339.60 ± 66.30, 161.40 ± 18.20 and 260.10 ± 43.70 mL/h/kg. AUC(0-∞) values of M2ES were 909.30 ± 199.60, 6251.00 ± 739.60 and 11758.00 ± 2010.10 ng∙h/mL. The dosage was positively correlated with AUC, and the correlation coefficient r2 = 0.9327. Self-developed ELISA assay could meet the requirements of serum M2ES concentration detection. PEGylation modification substantially expands the circulation time of recombinant human endostatin and effectively improves its pharmacokinetic behavior.