Aberrant alterations in genomic 5-hydroxymethylcytosine (5hmC), an oxidation product of 5-methylcytosine (5mC) by Ten-eleven translocation (TET) enzymes, are frequently associated with cancers. Quick and precise 5hmC quantification is vital since it is a key biomarker for diagnosis, pathophysiology, and therapy. Here, we present a portable, wireless potentiostat sensor for real-time, ultrasensitive 5hmC-DNA sensing based on a tree-like graphene (teG)-modified screen-printed microelectrode. One-pot electrochemical exfoliation of pencil graphite enabled the cost-effective, eco-friendly, and scalable synthesis of teG, which exhibited high electrical conductivity, excellent electrochemical conductivity, low surface roughness, and high 5hmC-DNA adsorption, surpassing those of pencil graphite (pG) and graphene oxide (GO). The teG-modified gold electrodes exhibited exceptional sensitivity (6.15 × 10-6 mM-1 cm-2), selectivity, and reproducibility, with an ultralow detection limit of 12.6 fM for 5hmC-DNA. The sensor’s performance was validated by quantifying 5hmC levels in genomic DNA from various biological specimens, including primary mouse tissues with altered TET function, mouse hepatocellular carcinoma, and human prostate cancer cell lines. To enhance practicality, a flexible, screen-printed microelectrode on mulberry paper was developed and integrated with a portable, wireless potentiostat powered by the Arduino Nano 33 IoT. Open-circuit potential (OCP)-based detection enabled label-free, real-time monitoring with wireless data transmission to an Android mobile application, successfully differentiating 5hmC levels between cancerous and noncancerous cells. These findings highlight teG’s high surface area, superior charge transport, and scalability, positioning it as a promising platform for next-generation biosensing. The developed sensor provides a rapid, cost-effective, and highly sensitive tool for 5hmC quantification, with significant implications for early cancer diagnostics and treatment.