Human parvovirus B19 is a prevalent childhood infectious virus that poses a great challenge to public health, so the detection of B19V is of great importance. In this study, a DNA sensor based on CbAgo, a Cas effector, and a dual electrochemical signal amplification strategy was developed by using a novel nanocomposite MnO2/CMK-3/g-C3N4/AgNPs for initial signal amplification, with CMK being an ordered mesoporous carbon nanomaterial. Single-walled carbon nanotubes (SWCNTs) were used as electrocatalytic probes for secondary signal amplification to detect B19 DNA. The detection process begins with polymerase chain reaction (PCR) amplification using the B19V infectious clone plasmid (pB19-M20) as a template and NS1-F/R as primers, followed by specific cleavage of B19 DNA based on the programmable cutting sites of CbAgo effector protein. This study enriches the application of Argonaute proteins in sensing and introduces a novel method to detect B19V. Under optimized conditions, the biosensor can detect B19 DNA in the range of 10−15–10−10 M, with a detection limit (LOD) of 0.2 fM. The results indicate that the developed DNA sensor holds promise for reliable and sensitive detection of B19 DNA in human serum.