Enzyme amplification is most commonly used to develop electrochemical affinity biosensors. However, single amplification by enzyme labels is not sufficient for detecting an ultra-low analyte concentration. Moreover, some of enzymatic products, such as thiocholine and hydroquinone (HQ), show poor electrochemical signals and/or are unstable in air for long incubation periods. In this work, we reported two "outer-sphere to inner-sphere" electrochemical-chemical-chemical (ECC) redox cycling reactions triggered by thiocholine and HQ. By examining the effect of different redox mediators and reductants, we found that ferrocenecarboxylic acid (FcA) and tris(2-carboxyethyl)phosphine (TCEP) were the optimal redox mediator and reductant, respectively. In the redox cycling, FcA was regenerated by thiocholine or HQ after its electro-oxidization; thiocholine or HQ was also then regenerated by TCEP after its oxidation, thus enhancing the anodic current of FcA. To demonstrate the applications and analytical merits of the two redox cycling reactions in biosensing, thrombin was tested in a "sandwich" format with acetylcholinesterase (AChE) and alkaline phosphatase (ALP) as the enzyme labels. The detection limits were determined to be 5 ngL(-1) and 0.5 ng L-1 with the thiocholine- and HQ-triggered redox cycling, respectively. The results will be valuable for developing enzyme-amplified electrochemical affinity biosensors. (C) 2014 Elsevier Ltd. All rights reserved.