The invention provides a pharmacokinetic testing method of Exendin-4, belonging to the analysis technical field. The testing method comprises the following steps: extracting and concentrating a human plasma sample by a solid phase extraction column, then diluting the concentrated sample into proper concentration, and detecting the Exendin-4 by a high performance liquid chromatography (HPLC)-ultraviolet detector; preparing solution containing 0.05mg/mL of the Exendin-4 by using acetonitrile-deionized water on the mass concentration of which is 85%, and taking the solution as W contrast; adding50muL of acetonitrile-deionized water the mass concentration of which is 85% to dissolve 0.5mL of the plasma sample after solid-phase extraction and lyophilization, and taking the dissolved solution as a tested sample W; and testing the chromatographic peak area of the obtained standard products as S contrast respectively and the chromatographic peak area of the tested sample as a S sample by HPLC, and finally calculating the content of the Exendin-4 in the tested sample and the content of the Exendin-4 in plasma by an external standard method respectively as follows: the W sample=(the S sample/the S contrast) the W contrast, and the W sample*50/0.5*106pg/mL. The testing method of the invention can analyze the content of the Exendin-4 in the plasma by the HPLC, thus being simple, convenient and reliable, and providing valid experimental data for clinical medication.