Bladder cancer is a common human malignancy. Conventional ultrasound and white-light cystoscopy are often used for bladder cancer diagnosis and resection, but insufficient specificity results in a high bladder cancer recurrence rate. New strategies for the diagnosis and resection of bladder cancer are needed. In this study, we developed a highly specific peptide-based probe for bladder cancer photoacoustic imaging (PAI) diagnosis and near-infrared (NIR)-imaging-guided resection after instillation. A bladder cancer-specific peptide (PLSWT7) was selected by in vivo phage-display technology and labeled with IRDye800CW to synthesize a bladder cancer-specific dual-modality imaging (DMI) probe (PLSWT7-DMI). The feasibility of PLSWT7-DMI-based dual-modality PAI-NIR imaging was assessed in vitro, in mouse models, and ex vivo human bladders. An air-pouch bladder cancer (APBC) model suitable for probe instillation was established to evaluate the probe-based bladder cancer PAI diagnosis and NIR-imaging-guided resection. Human bladders were used to assess whether the PLSWT7-DMI-based DMI strategy is a translatable approach for bladder cancer detection and resection. The probe exhibited excellent selectivity and specificity both in vitro and in vivo. Postinstillation of the probe, tumors <3 mm were detectable by PAI, and NIR-imaging-guided tumor resection decreased the bladder cancer recurrence rate by 90% and increased the survival in the mouse model. Additionally, ex vivo NIR imaging of human bladders indicated that PLSWT7-DMI-based imaging would potentially allow precise resection of bladder cancer in clinical settings. This PLSWT7-DMI-based DMI strategy was a translatable approach for bladder cancer diagnosis and resection and could potentially lower the bladder cancer recurrence rate. (C) 2018 AACR.