Topical photodynamic therapy (TPDT) is a clinical treatment for cutaneous squamous cell carcinoma (CSCC). However, the therapeutic efficacy of TPDT for CSCC is significantly weakened by hypoxia, which is caused by the oxygen-poor environment of the skin and CSCC and by the high oxygen consumption of PDT itself. To overcome these problems, we developed a topically applied perfluorotripropylamine-based oxygenated emulsion gel loaded with the photosensitizer 5-ALA (5-ALA-PBOEG) by a simple ultrasound-assisted emulsion method. With the aid of the microneedle roller, 5-ALA-PBOEG dramatically increased the accumulation of 5-ALA in the epidermis and the dermis, as well as throughout the dermis; a total of 67.6% ± 9.97% of the applied dose penetrated into and through the dermis, which is 19.1±3.2-fold that of the 5-ALA-PBOEG without microneedle treatment group, and 16.9±0.3-fold that of the aminolevulinic acid hydrochloride topical powder treatment group (p<0.001). Meanwhile, PBOEG enhanced the singlet oxygen yield of 5-ALA-induced protoporphyrin IX. The results of in vivo antitumor activity in human epidermoid carcinoma (A431) bearing mice showed that by increasing the oxygen content in tumor tissues, the developed 5-ALA-PBOEG plus microneedle treatment and laser irradiation showed better tumor growth inhibition than the respective control formulations. In addition, the results of safety studies, including the multiple-dose skin irritation study, allergy tests, and skin H&E staining, demonstrated the safety of 5-ALA-PBOEG plus microneedle treatment. In conclusion, the 5-ALA-PBOEG plus microneedle treatment shows great potential in the fight against CSCC and other skin cancers.Copyright © 2023. Published by Elsevier B.V.