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• A new nanoprobe (DTAAT) has been designed based on DNA tetrahedral supported hybridization chain reaction (HCR) for in situ analysis of bacteria. • DTAAT can recognize bacteria by dual binding of antibody and aptamer, which improves specificity and sensitivity for in situ analysis of bacteria. • DTAAT as a nanoprobe can be used for in situ imaging and quantitative analysis of bacteria. A new nanoprobe (DTAAT) has been well designed based on DNA tetrahedron supported hybridization chain reaction (HCR) and further applied for in situ analysis of bacteria. Antibody-aptamer conjugate (AA) has been coupled with DNA tetrahedron (DT) followed by the linkage of trigger strand (A). Bacteria can be preciously recognized by dual binding of both antibody and aptamer in DTAAT, to release trigger strand which is complementary with aptamer. By virtue of proximity effect, the released trigger strand can further efficiently induce the simultaneous occurrence of arrays of HCR at three vertices of DT, with rapid reaction rate of 359.29 min−1 in comparison with that of 212.60 min−1 for free HCR. DTAAT can be served for in situ imaging of bacteria by confocal laser scanning microscopy and single-cell characterization by flow cytometry. Moreover, using DTAAT as probe, the quantitative analysis of bacteria can be realized with high specificity and anti-interference capability. In a word, the developed DTAAT nanoprobe has a great potential for accurate and sensitive in situ analysis of bacteria. [ABSTRACT FROM AUTHOR]