Steady and efficient sensitized emission of Eu 2+ to Eu 3+ can be achieved through a rare mixed-valence Eu-MOF (L 4 Eu III 2 Eu II ). Compared with the sensitization of other substances, the similar ion radius and configuration of the extranuclear electron between Eu 2+ and Eu 3+ make sensitization easier and more efficient. The sensitization of Eu 2+ to Eu 3+ is of great assistance for the selfenhanced luminescence of L 4 Eu III 2 Eu II , the longer luminous time, and the more stable electrochemiluminescence (ECL) signal. Simultaneously, L 4 Eu III 2 Eu II possesses near-infrared (NIR) fluorescence of around 900 nm and a mighty selfluminous characteristic, which render it useful as a NIR fluorescent probe and as a luminophore to establish a NIR ECL biosensor. This NIR biosensor can greatly reduce the damage to the detected samples and even achieve a nondestructive test and improve the detection sensitivity by virtue of strong susceptibility and environmental suitability of NIR. In addition, the CeO 2 @Co 3 O 4 triple-shelled microspheres further enhanced the ECL intensity due to two redox pairs of Ce 3+ / Ce 4+ and Co 2+ /Co 3+ . The NIR ECL biosensor based on these strategies owns an ultrasensitive detection ability of CYFRA 21-1 with a low limit of detection of 1.70 fg/mL and also provides a novel idea for the construction of a highly effective nondestructive immunodetection biosensor.