En bloc resection of bladder tumor (ERBT) involves removing bladder tumors and their base. Laser resection has been used to reduce complications including bleeding and obturator nerve reflex (ONR). We developed a novel approach (rotatable bi-channel en bloc resection of bladder tumor (RBC-ERBT)) and assessed its efficacy in a pilot in-vivo study to enhance laser ERBT's applicability in challenging bladder regions. In the laser RBC-ERBT procedure, lesions were excised by inserting a holmium laser through the rotating external working channel, while forceps were inserted through the internal working channel provided traction on the tissue. Fifteen laser RBC-ERBT procedures were performed in five different bladder areas of three live pigs. The technical success rate (TSR), procedure time, lesion size, occurrence of complications (bleeding, perforation, ONR), and detrusor muscle (DM) presence rate and DM thickness were evaluated. All 15 procedures were performed with a 100% TSR. The resections were successful in all bladder regions (posterior, left, right and anterior walls and dome). Median procedure time was 20min. The resected specimen size was 10mm × 7mm to 17mm × 13mm. Mild bleeding occurred in two procedures (13.3%) but was effectively managed. No incidents of ONR or perforation were observed. Histological examination confirmed presence of DM in all specimens with a median DM thickness of 1.26mm. Our pilot in-vivo study suggested the feasibility and effectiveness of laser RBC-ERBT for bladder tumors in various locations. This technique offers effective traction, improved visualization, and enhanced laser accessibility. Further studies are required to validate its effectiveness in humans.