Hematoporphyrine injection (HpD)-based photodynamic therapy (HpD-PDT) has emerged as a promising cancer therapy. However, its tumor-targeting ability and metabolokinetics in nonmelanoma skin cancer (NMSC) have not been well explored. Importantly, photodynamic diagnosis is widely used for cancer lesion assessment and positioning to ensure effective therapy, while the photosensitizer metabolic kinetics study is utilized for biosafety assessment and light-protection instruction. These are particularly important for the optimization of therapeutic parameters. In the present study, NMSC patients were subjected to twice laser irradiation-based HpD-PDT strategy. Broadly, the study aimed to assess long-term variations in fluorescence (FL) intensity in vivo in NMSC patients after intravenous (i.v.) administration of HpD, and thus obtain information regarding metabolism, biosafety, and light-protection instruction for HpD during the therapy. In vitro experiments were used for the evaluation of absorption and fluorescent characterization of HpD in aqueous solution and cutaneous squamous cell carcinoma (SCC) cells. For in vivo assessment, 20 patients with NMSC, including SCC, basal cell carcinoma (BCC), Bowen disease (BD), extramammary Paget's disease (EMPD), and malignant proliferating tricholemmoma (APT), were recruited, and treated with HpD-PDT. To evaluate the selectivity and pharmacokinetics of HpD in vivo, relative changes in FL intensity for lesional, perilesional, and nonlesional skin of nonmelanoma skin cancer patients, before and after HpD injection, were semiquantitatively analyzed for 1month, using the FL detection system and Wood's lamp. The absorption and FL spectra were detected and semiquantitatively analyzed in HpD diluted solution and SCC cells after coincubation with HpD. After i.v. administration of HpD in EMPD patients, FL was detected in the skin lesions at 24 hours, and it was characterized by clear edges. Importantly, FL intensity in the skin lesions increased significantly at 48and 72hourspostinjection, which was suitable for HpD-PDT. After 72 h, it decreased gradually and reached close to the baseline value at 4 weeks postinjection. No severe side effects were observed during HpD injection and the therapy. Urinary tract infection was recorded in one patient (with a previous history of recurrent urinary tract infections) after HpD-PDT, and the patient was cured afterward. Transient light was observed in two patients after HpD-PDT and they soon recovered after therapy. The present study reported a significant increase in FL intensities at 48and 72hoursafter i.v. administration of HpD in patients with nonmelanoma skin cancers, which indicated accumulation of HpD at the cancer site. Importantly, HpD was found to be safe for NMSC patients. After therapy, FL intensities decreased, which indicated expending and metabolization of HpD. Thus, the results of the present study highlighted the suitability of a twice red-light laser irradiation strategy for the application of HpD-PDT in nonmelanoma skin cancer treatment.