Objective: This study focused on optimizing the Iota carrageenan extraction process and improving Iota carrageenan yield. Methods: Iota carrageenan was extracted from Eucheuma spinulosa by ultrasonic-assisted complex enzyme technology. Through single factor and orthogonal experiments, the optimum ratio of complex enzymes (cellulase, hemicellulose and papain) and enzymatic hydrolysis conditions of complex enzymes were first determined. Then, the optimum ultrasonic treatment conditions were determined. Finally, the boiling process conditions were optimized. Results: The optimal compound enzyme ratios were cellulase of 0.50%, hemicellulase of 1.25% and papain of 0.20%. The optimal enzymolysis conditions were as follows: enzymolysis at 60 ℃ and pH 5.5 for 2.5 h, with enzymolysis material-liquid ratio at 1∶30 (g/mL). The best ultrasonic treatment process was ultrasonic power of 350 W, treated at 40 ℃ for 25 min. The best boiling process was using 0.04% sodium hexametaphosphate (SHMP), boiling at 90 ℃ for 3 h. Conclusion: Under the control of the optimal process conditions, Iota carrageenan can reach a high extraction yield as 47.17%.