The development of biosensors for biologically important substances with ultralow content such as microRNA is of great significance. Herein, a novel surface plasmon-enhanced electrogenerated chemiluminescence-based aptasensor was developed for ultrasensitive sensing of microRNA by using nitrogen vacancy-rich carbon nitride nanosheets as effective luminophores and gold nanoparticles as plasmonic sources. The introduction of nitrogen vacancies improved the electrochemiluminescence behavior due to improved conductance and electrogenerated chemiluminescence activity. The introduction of plasmonic gold nanoparticles increased the electrochemiluminescence signal intensity by more than eightfold. The developed surface plasmon-enhanced electrogenerated chemiluminescence aptasensor exhibited good selectivity, ultrasensitivity, excellent stability, and reproducibility for the determination of microRNA-133a, with a dynamic linear range of 1 aM to 100 pM and a limit of detection about 0.87 aM. Moreover, the surface plasmon-enhanced electrogenerated chemiluminescence sensor obtained a good recovery when detecting the content of microRNA in actual serum.