Fluorescent probe-based analytical methods for biological species have gained increasing attention for their powerful detection and imaging capabilities. However, it is still occasionally problematic for accurate analyses due to the influence of the probe concentration, intrinsic fluorescence, instrumental factors or environmental conditions, and intense laser irradiation is usually harmful to biological systems. An NIR ratiometric fluorescent probe can eliminate the influence of these factors and lead to a higher sensing performance. We propose the introduction of a reasonable masking group onto the meso oxygen atom of a cyanine framework (Cy-O) to achieve a NIR ratiometric probe by modulating the conjugated polymethine π-electron system of the cyanine dye. Herein, we report a proof-of-the-concept attempt for a highly sensitive and selective mito-specific NIR ratiometric fluorescent sensing strategy for ALP, and the phosphate group was chosen as the functional group for ALP catalyzed hydrolysis. Upon treatment with ALP, the probe Cy-OP exhibits excellent recognition properties such as high selectivity, high sensitivity (with a low detection limit of 0.16 mU mL⁻¹), short response time (within 30 min) and a large spectral shift (from 736 to 516 nm in the absorption spectra and 766 to 616 nm in the emission spectra) with a low fluorescence background. Moreover, the ratio of emission (I_616nm/I_766nm) of the enzymatic residue was favorable for its application in a complex bio-system. Consequently, the well-designed probe was successfully applied for the detection of ALP in the living cells with satisfactory results.