The pH-responsive ultrasensitive fluorescent nanoprobe is composed of pH-responsive matrix materials and fluorescent organic small molecule dyes. The pH-responsive matrix materials are calcium phosphate, calcium hydroxyphosphate, fluorapatite, calcium carbonate or ZIF series; the fluorescent organic small molecule dyes are positively charged dyes or negatively charged dyes. The preparation method includes: coating a positively charged dye with a negatively charged matrix material; coating a negatively charged dye with a negatively charged matrix material; and coating a negatively charged dye with a positively charged matrix material. Compared with traditional small molecule fluorescent dyes, the present invention can greatly improve the sensitivity and specificity of fluorescence imaging and achieve ultrasensitive detection of tumor microenvironment response; the specific response probe prepared by the unique properties of the tumor microenvironment has the advantages of high targeting efficacy, low background signal, and high signal-to-noise ratio, and can achieve ultrasensitive detection of tiny tumors.