A new water soluble 2,2-bifunctionalized spin label (2,2E-BSL) having pyrrolidine nitroxide moiety was synthesized starting from a nitro compound with two hydroxymethyl group converted to the linkers of the 2,2E-BSL, and was applied to label troponin C (TnC). Labeled TnC through two linkages were successfully isolated, and 2,2E-BSL was proved to be immobilized on TnC by EPR measurement.