Abstract: Adeno-associated virus （AAV） is an efficient and safe gene delivery vector widely used in gene therapy and an indispensable tool in modern basic science. To reduce the cost of producing high-purity and high titer AAV vectors, we selected nanobodies （Nb） with high affinity for AAV as ligands, and an E. coli expression system was constructed to express the Nb. After purification with Ni Purose 6 Fast Flow, recombinant Nbs were obtained and coupled with Rigose SulfoLink preactivation medium to obtain affinity chromatography medium. The results showed that the prepared medium coupled with nanobodies had a concentration of 6.17 mg·mL⁻¹, with an adsorption loading of 1.92×10¹² VP·mL⁻¹ for AAV serotype 5. The adsorption loading of AAV1 and AAV7 reached the level of 1×10¹¹ VP·mL⁻¹ medium, both slightly higher than the commercial medium, and also had high adsorption loading for other serotypes. After 20 cycles of repeated use, the reusability of the homemade medium is comparable to that of the commercial medium, indicating good reusability. Using rAAV5-hRORA clinical grade gene therapy vector as a sample, the self-made medium showed a capture rate of not less than 60% and purity of 91% for the target virus.