Antifreeze protein from Pseudopleuronectes americanus inhibits ice crystal growth and regulates morphology, demonstrating significant application potential in food cryopreservation. However, its low heterologous expression level and soluble expression yield hinder industrial application. To enhance its expression level and soluble expression, an antifreeze protein-engineered strain EcN-pET30-E-(LA)3 expressing a fusion enhanced green fluorescent protein was employed as the research subject. The antifreeze protective properties of the fusion protein were investigated, a detection method was established, and fermentation conditions were optimized. Results demonstrated that the fusion protein was successfully expressed visually. The survival rate of the strain after treatment with this protein was 24.35%, retaining 90.35% of that prior to fusion. The fluorescence standard curve was Y=135.40X?16.99 (X: fusion protein concentration, µg·mL-1; Y: fluorescence intensity, A.U.), exhibiting good linearity (R2=0.9981). Single-factor optimization determined the optimal fermentation conditions: supplementation of 25 mmol·L-1 K?HPO? in LB medium, addition of 1 mmol·L-1 IPTG and 15 mmol·L-1 glycine after 5 h of cultivation, supplementation of 0.2% glucose after 2 h of induction at 20 °C, and continued induction to 20 h, achieving a soluble fusion protein yield of 0.70 mg·mL-1, which was 3.8-fold that before optimization, accounting for 36.91% of intracellular soluble proteins, representing a 75.76% increase compared to pre-optimization levels. These results indicated that the fusion expression did not significantly affect the antifreeze properties of the antifreeze protein., the fluorescence method is rapid and accurate, and fermentation condition optimization significantly enhanced the expression level and soluble expression proportion of the target protein, providing a theoretical foundation for the efficient preparation of antifreeze proteins.