One of the critical problems in bladder cancer (BC) management is local recurrence of disease. However, achieving accurate delineation of tumor margins intraoperatively remains extremely difficult due to the lack of effective tumor margin recognition technology. Herein, survivin molecular beacon (MB) and R11 peptides-linked spherical mucleic acids (SNAs) were synthesized as a nanoprobe (AuNP-MB@R11) for sensitive detection of BC margins. Physicochemical Properties proved that R11 peptides and survivin MB were successfully loaded onto the surface of SNAs. The AuNP-MB@R11 had good stability against nuclease, high sensitivity and specificity to detect survivin ssDNA in vitro experiment. In cytology, R11 peptides could increase BC targeting ability and membrane penetrability of SNAs. Notably, R11 peptides can significantly promote the disintegration of lysosome and release of SNAs to enhance the fluorescence imaging quality. The further RNA sequencing proved that some genes related with endocytosis and lysosome regulated significantly, such as AGPAT5, GPD1L, TREX1 and APEX1. In orthotopic BC models and a clinical sample from patient with BC, AuNP-MB@R11 showed a more legible cancerous fluorescence margin and offered remarkably improved detection effects compared to those achieved by SNAs. Thus, R11 peptides-linked SNAs presented a promising potential to identify the BC margin, which may help to improve the R0 resection rate in surgery and improve patient’s quality of life.