Background and purpose: Actinic keratosis (AK) is a precancerous condition with the potential to develop into cutaneous squamous cell carcinoma. 5-Aminolevulinic acid photodynamic therapy (ALA-PDT) has emerged as a new treatment option for AK due to its high clearance rates and non-invasive cosmetic advantages. However, its efficacy in treating Olsen grade 3 AK, characterized by hyperkeratosis, remains controversial. This study aimed to investigate the efficacy and safety of ALA-PDT in treating Olsen grade 3 AK and to identify factors influencing treatment outcomes. Methods: A total of 38 patients with Olsen grade 3 AK who visited the Department of Dermatology, Huashan Hospital, Fudan University, between January 2020 and July 2023 underwent four consecutive sessions of ALA-PDT and were followed up for one year post-treatment (ethics number: 2019-491). All patients met the inclusion and exclusion criteria. Treatment efficacy was assessed by the initial complete clearance (ICC) at 3 months and the sustained complete clearance (SCC) at 12 months after treatment. Baseline clinical and pathological characteristics were collected. Univariate and multivariate logistic regression analyses were performed to explore risk factors for treatment failure in Olsen grade 3 AK patients receiving ALA-PDT. Subgroup analyses were conducted to further investigate risk factors associated with treatment resistance and recurrence. This study was registered on Chinese Clinical Trial Registry (chiCTR1800019213). The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist was followed for this study. Results: A total of 38 patients were included in this study, including 8 in the case group and 29 in the control group. One patient was lost to follow-up 6 months after treatment. At 3 months post-treatment, 86.84% of patients achieved ICC (33/38). At the 12-month follow-up, 87.88% of the patients who achieved ICC maintained SCC (29/33). No serious adverse reactions were reported during treatment and follow-up. Multivariate logistic regression analysis indicated that lesions located in multiple anatomical subunits was an independent risk factor for treatment failure (P=0.02, OR=28.43). Subgroup analysis confirmed that this factor was independently associated with treatment resistance (P=0.03, OR=97.54). Conclusion: ALA-PDT is effective and safe for treating Olsen grade 3 AK, offering a non-invasive treatment option for these patients. However, patients with lesions located in multiple anatomical subunits are more prone to treatment failure, warranting increased clinical attention in this population.