We have devised a barcode DNA-mediated signal amplifying strategy for ultrasensitive biomolecular detection by utilizing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). As a model target, thrombin was first captured by specific aptamer15 functionalized on magnetic beads (MBs-apt15) and sandwiched through the simultaneous interaction with gold nanoparticles modified with another specific aptamer29 and barcode DNA molecules (apt29-AuNPs-bcDNAs). The sandwiched complex was collected by convenient magnetic separation and then treated with potassium cyanide (KCN) to dissolve the gold nanoparticles (AuNPs) and consequently release the barcode DNA molecules (bcDNAs), which were then again magnetically separated and analyzed by using MALDI-TOF MS. Under optimized conditions, this strategy revealed an excellent sensitivity with a limit of detection of 0.89 aM in a wide linear detection range from 0 aM to 0.1 nM and exhibited an acceptable recovery for thrombin detection in complex biological matrices. This signal amplifying strategy based on MALDI-TOF MS could greatly enable the ultrasensitive detection of various low abundant biomolecules.