An ingenious microtubule functionalization strategy based on the novel bioorthogonal conjugation is developed to construct fluorescent ratiometric probes for ATP sensing. By single excitation, newly synthesized probe RT-1 exhibited green emission (485 nm) from the bioorthogonal fluorescence conjugation part and orange emission (584 nm) from the ATP sensing part. The fluorescence intensity ratio (I₅₈₄/I₄₈₅) displayed good linear response in ATP ranges of 0–2.5 mM (y = 0.9282x + 0.2398, R² = 0.9705, LOD = 0.0354 mM) and 2.0–10.0 mM (y = 2.7153x-3.6234, R² = 0.9911, LOD = 0.0121 mM). Moreover, a paclitaxel derivate probe RT-2 was developed to functionalize in vitro polymerized microtubule for in situ ATP detection. This simple and convenient strategy is anticipated to stimulate more microtubule related applications in both in vitro and intracellular studies.