This paper reports a novel integrated circuit for fluorescence sensing. The circuit implements a differential readout architecture in order to reduce the overall noise figure. The circuit has been fabricated in a commercially available 0.5 μ m CMOS technology. Preliminary results show that the reset noise is reduced by a factor of 1.42 and the readout noise by a factor of 9.20 when the pixel is operated in differential mode versus single-ended mode. Spectral responsivity characteristics show that the photodiodes are most sensitive at 480 nm. Using a commercially available emission filter, the sensor was able to reliably detect a concentration of Fura-2 as low as 39 nM. The sensor was used to perform ratiometric measurements and was able to reliably detect a free calcium concentration of 17 nM.