Fluorescent gold nanoclusters (AuNCs) were incorporated into porous calcium carbonate spheres through electrostatic interaction. The resulting CaCO(3)/AuNCs hybrid material exhibited interesting properties, such as porous structure, excellent biocompatibility, good water solubility, and degradability. These properties make the CaCO(3)/AuNCs hybrid material a promising template to assemble horseradish peroxidase/antibody conjugates (HRP-Ab(2)). By using CaCO(3)/AuNCs/HRP-Ab(2) bioconjugates as probes, a versatile immunosensor was developed for fluorescent and electrochemical detection of the cancer biomarker neuron-specific enolase (NSE). The detection limits of the sensor were 2.0 and 0.1 pg mL(-1) for fluorescent and electrochemical detection, respectively. The immunosensor shows high sensitivity and offers an alternative strategy for the detection of other proteins and DNA.