We propose and demonstrate a novel temperature-insensitive bio-sensor for accurate and quantitative detection of Escherichia coli (E. coli) bacteria in water. Surface sensitivity is maximized by operating the long-period fiber grating (LPFG) closest to its turnaround wavelength, and the temperature insensitivity is achieved by selectively exciting a pair of cladding modes with opposite dispersion characteristics. Our sensor shows a nominal temperature sensitivity of ∼1.25 pm/°C, which can be further reduced by properly adjusting the LPFG lengths, while maintaining a high refractive index sensitivity of 1929 nm/RIU. The overall length of the sensor is ∼3.6 cm, making it ideally suitable for bio-sensing applications. As an example, we also show the sensor's capability for reliable, quantitative detection of E. coli bacteria in water over a temperature fluctuation of room temperature to 40°C.