Herein, π-conjugated polypyrene network (CPN) was synthesized by Suzuki-Miyaura polycondensation that exhibited high electrochemiluminescence (ECL) emission. The cross-linked network structure eliminates the aggregation-induced quenching effect of pyrene through steric hindrance. On the one hand, the extended π-conjugated backbone facilitated the acceleration of charge transfer and improved the stability of free radicals in the redox process, and on the other hand, by virtue of the porous structure, the coreactant and electrolyte could diffuse rapidly and fully penetrate into the polymer interior, thus effectively improving the ECL efficiency of the CPN. In view of the excellent ECL performance, an ECL biosensor for the ultrasensitive detection of the lung cancer biomarker microRNA let-7a was constructed by using CPN as a novel ECL probe in combination with a target-triggered rolling circle amplification strategy that exhibited a linear range from 1 aM to 10 pM with a low detection limit of 0.33 aM. In a word, this work provides an effective method to improve the ECL strength of polymers and opens up new prospects for developing new ECL emitters.