Since the lack of detection diversity of the single-signal readout strategy, it is urgent to develop fast and multisignal assay strategies. A highly selective and sensitive assay method with colorimetric and fluorometric dual signals readouts is presented in this paper. It is based on the principle that the target induced conjunction of split aptamer fragments assembled on the surface of Au nanoparticles (AuNPs). In the presence of targets, the color of solution changed from wine red to blue and can be measured both visual inspection and spectrophotometry because of the aggregation of AuNPs. At the same time, the report probes which are original hybrid with the anchoring aptamer fragments on the AuNPs surface can be released and recovers the fluorescence. By use of this detection strategy, the limit of detection for thrombin (TMB), as a model of analyte, were 0.45 and 0.16nM, respectively. Furthermore, this protocol can discriminate TMB from other analogue with high selectivity and can be used to detect TMB in human serum samples. The results came from the two signals were well consistent with each other, which demonstrated that it has application potential for detection of TMB in complex matrix.