A new fluorescent and colorimetric Cu2+ probe was synthesized, and realized optical imaging in RAW264.7 macrophages. The design strategy was based on a change in structure between spirocyclic (non-fluorescent) and ring-open (fluorescent) forms of rhodamine-based dyes, and its crystal structure was presented to explain the binding mode. Upon treatment with Cu2+, the weakly fluorescent probe exhibited a strong fluorescence response, high selectivity and was quantitative for Cu2+ under physiological conditions. In addition, the off-on-type fluorescent change upon the addition of Cu2+ was also applied in bioimaging.