Glucose and ATP biosensors have important applications in diagnostics and research. Biosensors based on conventional materials suffer from low sensitivity and low spatial resolution. Our previous work has shown that combining single-walled carbon nanotubes (SWCNTs) with Pt nanoparticles can significantly enhance the performance of electrochemical biosensors. The immobilization of SWCNTs on biosensors remains challenging due to the aqueous insolubility originating from van der Waals forces. In this study, we used single-stranded DNA (ssDNA) to modify SWCNTs to increase solubility in water. This allowed us to explore new schemes of combining ssDNA-SWCNT and Pt black in aqueous media systems. The result is a nanocomposite with enhanced biosensor performance. The surface morphology, electroactive surface area, and electrocatalytic performance of different fabrication protocols were studied and compared. The ssDNA-SWCNT/Pt black nanocomposite constructed by a layered scheme proved most effective in terms of biosensor activity. The key feature of this protocol is the exploitation of ssDNA-SWCNTs as molecular templates for Pt black electrodeposition. The glucose and ATP microbiosensors fabricated on this platform exhibited high sensitivity (817.3 nA/mM and 45.6 nA/mM, respectively), wide linear range (up to 7 mMand 510 mM), low limit of detection (1 mM and 2 mM) and desirable selectivity. This work is significant to biosensor development because this is the first demonstration of ssDNA-SWCNT/Pt black nanocomposite as a platform for constructing both single-enzyme and multi-enzyme biosensors for physiological applications.