A series of Zr-based metal organic framework (MOF) composites embedded with three kinds of aptamer strands (509-MOF@Apt) were achieved by a one-step de novo synthetic approach. A platform for ultrasensitive detection of analytes, namely, thrombin, kanamycin, and carcinoembryonic antigen (CEA), was also established. Considering the conformational changes caused by the binding; interactions between aptamer strands and targeted molecules, the label-free electrochemical aptasensors based on 509-MOF@Apt composites could be developed to detect various target molecules. By comparing the common fabrication approaches of aptasensors, a distinct determination mechanism was presented through analysis of the electrochemical measurements on different interaction behaviors between probe aptamer strands and 509-MOF materials. The optimized aptasensors based on 509-MOFs@Apt demonstrated excellent sensitivity (with the detection limit of 0.40, 0.37, and 0.21 pg mL(-1) for CEA, thrombin, and kanamycin, respectively), stability, repeatability, and applicability. This work will provide a new platform for direct and feasible detection in biosensing related to clinical diagnostics and therapeutics, and further, extend the scope of potential applications for MOF materials.