Bladder cancer (BC) has a high recurrence rate, necessitating frequent monitoring. We herein present an innovative method for detecting BC-related miR-183 and miR-155 microRNAs using pH-responsive triplex DNA nanoswitches (TDNs). This approach employs a stepwise surface plasmon resonance biosensing platform (TDNs-SPR assay) to detect these two miRNAs sequentially. The platform involves the assembly of two triplex pH-responsive probes, switch A (SA) and switch B (SB), on an SPR sensing interface by anchoring the probes to the surface through SA/miR-183 and SB/miR-155 binding to the S9.6 antibody-modified surface. The probes are functionalized with streptavidin-Au nanoparticles/biotinylated strands, which act as reporter units for the presence of the respective miRNAs on the sensing interface. The pH-induced displacement of reporter units triggers stepwise SPR reflectivity changes: at pH 5.0 for sensing miR-183 and at pH 8.3 for sensing miR-155. The reflectivity changes relate quantitatively to the concentrations of miRNAs. This sensing platform enables the detection of two miRNAs with detection limits as low as 0.57 pM for miR-183 and 0.83 pM for miR-155, highlighting its powerful utility for precise biomarker analysis. Moreover, this platform distinguishes BC patients from healthy individuals in urine samples. The method offers a versatile, noninvasive method for detecting any two miRNAs associated with other diseases.