The excess residues of fluoride ions cause serious human health problems, making their detection highly valuable. In this work, a whole-cell-based biosensor was presented for the detection of fluoride ions, which can inhibit the color reaction of 3,3′,5,5′,-tetramethylbenzidine (TMB) catalyzed by the CotA-laccase of spore surface. This reaction for the detection of fluoride ions could be read out through UV–vis spectrophotometer, smartphone, or standard colorimetric card within 10 min. Under optimum conditions, a linear range of 1–600 umol L^–1 with a detection limit of 0.12 umol L^–1 (3o/k) was achieved for fluoride ions detection by using UV–vis spectrophotometer. The biosensor coupling with smartphone had a good linear response to fluoride ions concentration in the range of 5–600 umol L^–1 with LOD of 0.90 umol L^–1 (3o/k). The standard colorimetric card can be directly used for recognizing the fluoride ions level via naked-eyes. A portable kit based on a colorimetric card and smartphone was developed and has been successfully applied for fluoride ions monitoring in surface waters and groundwater. This developed method has several advantages such as rapid, outstanding selectivity and anti-interference, low-cost, ease of operation and storage, and eco-friendliness, meeting the demands of point-of-care testing of fluoride ions and disease prevention.