We synthesized a fluorescent probe (1) based on a novel receptor, phosphoserine for ratiometric detection of In(III). 1 selectively detected In(III) among various metal ions including Al(III) and Ga(III) in aqueous solutions through a ratiometric response. Upon addition of In(III), monomer emission of the pyrene at 395 nm decreased and the excimer emission at 476 nm increased significantly. About 2 equiv of In(III) completely induced the ratiometric response of 1. The detection limit was calculated to be 64 nM (R2 = 0.992) in aq. solution. Job’s plot analysis indicated that 1 formed a 2:1 complex with In(III) and the binding affinity of 1 for In(III) was measured to be 2.3 × 1012 M-2 (R2 = 0.989). The binding mode study showed that the phosphate group and two carbonyl groups of the amide bonds of 1 play an important role for the binding with In(III). Moreover, the fluorescent probe was suitable for the ratiometric detection of In(III) ions in ground water and tap water.