A novel hydrogen-bonded organic framework (HOF-14) has attracted much attention due to its excellent biocompatibility and low toxicity, but its research in the electrochemiluminescence (ECL) field has not been reported. In this work, the annihilation-type and coreactant-type ECL emission mechanisms of HOF-14 were studied systematically for the first time. It was found that the ECL quantum efficiency of HOF-14/TEA coreactant system was the highest, which was 1.82 times that of Ru(bpy) 3 2+ /TEA. Further, the ECL emission intensity of HOF-14/TEA system could achieve colorimetric (CL) imaging of mobile phone. We also discovered that HOF-14 had superior photoelectrochemical (PEC) performance. Based on the above research results, a unique HOF-14-based multimode sensing and imaging platform was constructed. The antibiotic Enrofloxacin (ENR) was selected as the detection target, and the Cu−Zn bimetallic single-atom nanozyme (Cu−Zn/ SAzyme) with excellent peroxidase (POD)-like activity was used to prepare quenching probes. When the target ENR was present, Cu−Zn/SAzyme quenching probes were introduced to the surface of HOF-14 by the dual-aptamer sandwich method. Cu−Zn/SAzyme could catalyze diaminobenzidine (DAB) to produce brown precipitations to quench the ECL, PEC, and CL signals of HOF-14, realizing multimode detection of ENR. This work not only discovered excellent ECL and PEC property of new HOF-14 material but also systematically studied the ECL emission mechanism of HOF-14, and proposed a novel multimode sensing and imaging platform, which greatly improved the detection accuracy of target and showed great contributions to the field of ECL analysis.