Two pyridine‐based isomeric fluorescent probes, 3‐(4‐dimethylamino‐phenyl)‐2‐pyridin‐3‐yl‐acrylonitrile (1) and 3‐(4‐dimethylamino‐phenyl)‐2‐pyridin‐4‐yl‐acrylonitrile (2), were successfully designed, synthesized and characterized with NMR, LC‐MS and HRMS methods. Both probes are pH‐reversible, showing an excellent selectivity toward H+ than other coexisting metal ions. They display good linear response within the extreme‐acidity range of 1.85–2.75 with the pKa of 2.28 for 1, and that of 2.65–4.17 with the pKa of 3.15 for 2, respectively. The probe 2 has the ratiometric fluorescence emission (I513nm/I570nm) characteristics and could be used as a sensor of acidity. The color of 2 is changed from yellow to pink, allowing colorimetric detection of pH by the naked eyes. In addition, both 1 and 2 could be used for monitoring pH in Escherichia coli (E. coli) cells under the extremely acidic conditions.