Abstract The development of sensitive and simple sensing strategies for trace proteins has received increasing attentions in the diagnosis and treatment of diseases. We describe here an electrode immobilization-free and homogeneous electrochemical method for sensitively detecting thrombin in human serums. The strategy relies on target-promoted proximity binding hybridization chain reaction (HCR) formation of magnetic nanoparticle (MNP)-decorated DNA polymers. Simultaneous binding of thrombin by two distinct aptamers increases their local concentrations and enables the proximity binding-induced strand displacement reaction, which leads to subsequent initiation of HCR between the methylene blue (MB)- and biotin-labeled hairpins into long DNA polymers. The streptavidin-modified MNPs further bind the biotin moieties to form the MNPs/DNA polymers. Subsequent accumulation of the MNPs/DNA polymers on the AuNP-deposited electrode can thus yield substantially enhanced current, due to the oxidation of the many MB labels, for highly sensitive detection of thrombin ranging from 5 pM∼50 nM with a detection limit of 1.1 pM in a simple electrode immobilization-free way. Selective interrogation of low levels of thrombin in diluted human serums was also verified, revealing its potential for convenient and ultrasensitive monitoring of a variety of protein biomarkers for disease diagnosis at the early stages.