Objective: To investigate the therapeutic effect of aminolevulinic acid photodynamic two-step irradiation on pain in patients with intraluminal condyloma acuminatum. Methods: 60 patients with intraluminal condyloma acuminatum treated with aminolevulinic acid photodynamic therapy (ALA-PDT) in the dermatology department of Xi’an Third Hospital from May 2019 to September 2021 were selected and randomly divided into the experimental group and the control group. The experimental group received aminolevulinic acid photodynamic two-step irradiation treatment while the control group was treated with conventional photodynamic irradiation. The experimental group and the control group were treated once every 7–10 days for four consecutive times. The treatment effect and pain score were recorded. Results: The efficacy of aminolevulinic acid photodynamic therapy was observed two weeks after the end of the treatment. The complete response rate of the experimental group was 66.7% (20/30), the partial response rate was 33.3% (10/30), and the ineffective rate was 0. In the control group, the complete response rate was 70% (21/30), the partial response rate was 30% (9/30), and the ineffective rate was 0. There was no significant difference in the complete response rate between the experimental group and the control group (χ² = 0.527, P = 0.706). Pain scores were evaluated at 5 minutes and 20 minutes during photodynamic therapy, and 30 minutes after photodynamic therapy. The pain scores of patients in the experimental group were lower than those in the control group at 5 minutes, 20 minutes, and 30 minutes after treatment. There was no significant difference in adverse reactions between the experimental group and the control group. Conclusion: Aminolevulinic acid photodynamic two-step irradiation can effectively ensure the therapeutic effect of patients with intraluminal condyloma acuminatum, and can significantly reduce patients’ pain during and after treatment.