Ratiometric electrochemiluminescence (ECL) assays have been widely applied in biosensing because of eliminated outside interferences and improved reliability in detection. In order to construct ratiometric ECL biosensors with high sensitivity and reliability, it is critical to find two signal emitters with suitable applied potentials or emission wavelengths. This review aims to discuss recent advances and trends of ratiometric ECL biosensors in terms of ECL materials and corresponding ratiometric sensing approaches. We focus on four types of ratiometric ECL biosensors based on particular ECL materials and ratiometric sensing strategies.