The functional fragment V_H/κ of an anti-tumor antibody AA98 was refolded in vitro by dilution refolding, gel filtration chromatography and urea gradient gel filtration, respectively. Concerning various factors in the renaturing buffer, such as GSH/GSSG ratio, arginine concentration, pH, flow rate, protein concentration in denaturing buffer and linear gradient, a urea gradient gel filtration method for V_H/κ refolding was established. As a result, compared with the traditional dilution refolding and gel filtration, the activity recovery of V_H/κ obtained by urea gradient gel filtration was significantly improved.