Spherical alginate beads were prepared by ionotropic gelation of sodium alginate through the use of calcium ions. Pyranine (Py) was added to the alginate solution as a small molecule probe for fluorescence studies. Desorption of Py in water from the alginate beads cross-linked with calcium ions was studied by using the steady state fluorescence technique. The fluorescence emission intensity (I) from Py was monitored during the desorption process at 512 nm using the time drive mode of the spectrofluorometer. The increase in I was attributed to Py release from the beads. The Fickian diffusion model was used to calculate the desorption coefficients, D, which were found to be increased up to 3% (w/v) CaCl2 concentration in the beads, and then decreased with a further increase of CaCl2 content. On the other hand, the encapsulation efficiency of Py in the calcium alginate beads presented the reverse behavior compared to D. It was observed that, when the content of CaCl2 was increased, the incubation time, t(0), for the start of desorption increased.