Poly(methylene blue) prepared by the widely used electrochemical polymerization method suffers from the undefined chemical structure and difficulty in modification with functional groups. The chemical polymerization of methylene blue is a challenging but attractive subject. We designed and prepared a methylene blue-backboned polymer (MBP-SO 3 ) with the side chain modified with sulfonic acid groups. The polymer retains the redox activity of methylene blue and has a good mass transfer effect in electro-catalyzing the oxidation of dopamine, epinephrine, and uric acid. The negative charges carried by MBP-SO 3 under physiological conditions are beneficial to attract cationic biomolecules, and its nanosheet-like morphology is conducive to charge transfer, which amplifies electrochemical signals in detection of target biomolecules. Using the nafion film coating the electrode surface to block ascorbic acid and nicotinamide adenine dinucleotide (NADH), the MBP-SO 3 modified electrode can selectively detect dopamine (0.02–25 mM), epinephrine (0.1–30 mM) and uric acid (0.03–3 mM) without interference from high-concentration of ascorbic acid and NADH solutions. The detections are also effective in serum samples. This work paves a way for methylene blue-backbone polymers in electrochemical sensing. • A sulfonated methylene blue-backboned polymer, MBP-SO 3 , was synthesized through oxidative polymerization. • The lamellar morphology of MBP-SO 3 provides a large number of active sites. • The sulfonate groups of MBP-SO 3 can promote enrichment of bio-analytes and proton transfer, while methylene blue units can facilitate electron transfer. • MBP-SO 3 is advantageous than conventional poly(methylene blue) in electrochemical detection of dopamine, epinephrine and uric acid with high sensitivity and stability. • The MBP-SO 3 @nafion electrode exhibits anti-pollution and anti-interference capability.