Direct electron transfer (DET) between a redox label and an electrode has been widely used for sensitive and selective sandwich-type detection without a wash step. However, it is still challenging to apply DET to protein detection, and a single redox label per probe is insufficient to obtain a high electrochemical signal. Here, a wash-free, sandwich-type method for thrombin detection based on DET and catalytic signal amplification by multiple redox labels, has been reported. The detection method involved (i) the redox label-catalyzed oxidation of a reductant, (ii) using a ploy-linker for the conjugation of multiple redox labels per probe (iii) the low nonspecific adsorption of the conjugated poly-linker due to uncharged, reduced redox labels, and (iv) a facile DET using long, flexible poly-linker and spacer DNA. Amine-reactive phenazine ethosulfate and NADH were used as the redox label and reductant, respectively. For the conjugation between an aptamer probe and multiple redox labels, N₃-terminated polylysine was used as the poly-linker. Approximately 11 redox labels per probe and rapid catalytic NADH oxidation contributes to high amplification of the signal. Thrombin in urine could be detected with a detection limit of ~50 pM without a wash step, which is practically promising for point-of-care testing of proteins.