Selective determination of aquatic chromium is critically important because of the dramatic differences in health and environment impacts by trivalent and hexavalent forms of chromium; however, it is challenging. In this work, for the first time, a nonconjugated polymer fluorophore (GCPF) was synthesized by cross-linking chitosan with glutaraldehyde via Schiff base reactions and systematically investigated for selective determination of Cr(VI). The results revealed that the synthesized GCPF exhibited excellent photostability and water solubility. More importantly, GCPF possessed dramatically enhanced fluorescence intensity originated from the n−π* transitions of the Schiff base subfluorophore groups (e.g., C═N) that can be selectively and sensitively quenched by Cr(VI) through oxidative damages to C═N group. An effective EDTA masking agent approach was employed to minimize ionic interferences. In the presence of high concentration of interference ions including Cr(III), the quenching GCPF fluorescence is capable of selectively determining Cr(VI) within a concentration range up to 50 μM and a detection limit of 0.22 μM. The analytical performance of GCPF was also confirmed by analyzing real surface water and industrial samples.