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Upconversion fluorescence has triggered extensive efforts in the past decade because of its superior physicochemical features and great potential in biomedical and biophotonic studies. However, practical applications of upconversion fluorescence are often hindered by its relatively low luminescence efficiency (<1%). Here, we employ a living yeast or human cell as a natural bio-microlens to enhance the upconversion fluorescence. The natural bio-microlens, which was stably trapped on a fiber probe, could concentrate the excitation light into a subwavelength region so that the upconversion fluorescence of core-shell NaYF 4 :Yb 3+ /Tm 3+ nanoparticles was enhanced by 2 orders of magnitude. As a benefit of the fluorescence enhancement, single-cell imaging and real-time detection of the labeled pathogenic bacteria, such as Escherichia coli and Staphylococcus aureus, were successfully achieved in the dark fields. This biocompatible, sensitive, and miniature approach could provide a promising powerful tool for biological imaging, biophotonic sensing, and single-cell analysis.