Rapid and low-cost diagnosis of multiple infectious diseases is of great significance especially in densely populated or resource-constrained settings. Herein, we developed a one-step fast and label-free imaging array for multiplexed detection of trace avian influenza virus (AIV) DNA biomarkers. By designing a series of specific and efficient catalytic hairpin assembly (CHA) amplification reactions and utilizing thioflavin T, a specific G-quadruplex fluorescence probe, three subtypes of AIV DNA biomarkers (H1N1, H7N9 and H5N1) were simultaneously and quickly detected within only 20 min, which just needed a small reagent volume of 50 μL and a smartphone instead of a spectrometer. With the combination of fluorescence imaging output and grey-level analysis, the array sensor can be on-site with the limit of detection of 136 pM, 141 pM and 129 pM for H1N1, H7N9 and H5N1, respectively. The imaging array also displayed good mismatch discrimination, excellent anti-interference, and real sample application. In view of its advantages of fast detection, low cost and multiplexed analysis, the imaging array is expected to have potential applications for early infectious disease diagnosis in resource-constrained settings.