In vitro diagnosis requires target biomarkers to be reliably detected at an ultralow level. A dual-signal strategy permits self-calibration to overcome the interferences of experimental and environmental factors, and thus is regarded as a promising approach. However, currently reported works mainly concentrated on the same forms of energy of output signals. Herein, we propose a one-step strategy for synthesis of methylene blue-encapsulated zeolitic imidazolate framework-90 (MB@ZIF-90) with high loading, unique dual-signal property, exceptional recognition capability, and good stability, and we further pioneer MB@ZIF-90 as a dual-signal biosensor for label-free, enzyme-free, and ultrasensitive detection of adenosine triphosphate (ATP) by integration of fluorescence and homogeneous electrochemical techniques. The recognition of MB@ZIF-90 by target ATP spurs the decomposition of ZIF-90, subsequently permitting MB to be released into a supernatant. As compared to the case where ATP does not exist, obviously increased intensities in fluorescence and differential pulse voltammetry current are observed and both signals are directly proportional to ATP concentrations. Thus, the MB@ZIF-90-based biosensor achieved dual-signal detection of ATP in an ultrasensitive manner and displayed a more reliable diagnosis result than previously reported ATP biosensors. This dual-signal strategy provides a new opportunity to develop high-performance biosensors for in vitro diagnosis and demonstrates great potential for future applications in bioinformatics and clinical medicine.