A paper-based chemiluminescence (CL) immunodevice is described for sensitive determination of the carcinoembryonic antigen (CEA). Capture antibody (Ab1) was first immobilized on the paper-based chip after plasma treatment of the paper surface. Next, carbon nanospheres functionalized with horse radish peroxidase (HRP) were attached as a signal antibody label. Strongly enhanced CL was achieved from the HRP catalyzed luminol/H2O2 CL system when using a sandwich immunoassay format. CEA can be quantified with a lower detection limit of 3 pg·mL−1. The method displays an almost 10-fold increased sensitivity in comparison to a commercial Ab2-HRP based kit. Conceivably, this sensitive method may also be extended by designing sensitive assays for other biomarkers of interest in point-of-care diagnostics. Graphical abstractSchematic of a paper-based chemiluminescence (CL) immunodevice for the determination of the carcinoembryonic antigen (CEA) by applying a multi-enzyme carbon nanosphere signal enhancement strategy.