In order to study the in vitro release pattern of copper triethylenetetramine (TETA-Cu) from copper-loaded nanoparticles in nasal in situ gels (TETA-Cu-NPs-gel). Dialysis was used to investigate in vitro release. 0.1 mol/mL phosphate buffer solution (PBS, pH = 7.4) was used as the release medium and the model was fitted to the release data using Origin software. HPLC (high performance liquid chromatography) was used to investigate the release level of TETA-Cu in dissolution media. The chromatographic column was Swell ChromplusTM C18 (4.6 mm × 150 mm, 5 μm), mobile phase A was pH = 3.0 phosphate buffer, and mobile phase B was methanol (A∶B=90 ∶ 10). The flow rate was 1.0 mL/min, the detection wavelength was 260 nm, Intake quantity 20 μL. The results show that TETA-Cu showed good linearity in the range of 0.05 ~ 1 mg/mL. The recoveries of the high, medium, and low concentrations are in the range of 99.9% ~ 101.0%, with the RSDs of less than 2%. Compared with the TETA-Cu solution, the rate of drug release of TETA-Cu-NPs-gel is slow, which conformed to the kinetic equation of the first level of drug release. It is concluded that the in vitro release assay of TETA-Cu-NPs-gel established in this study is accurate, simple, and feasible. TETA-Cu-NPs-gel has a slow-release effect.