The L-lysine fermentation process is a complex, nonlinear, dynamic biochemical reaction process with multiple inputs and multiple outputs. There is a complex nonlinear dynamic relationship between each state variable. Some key variables in the fermentation process that directly reflect the quality of the fermentation cannot be measured online in real-time which greatly limits the application of advanced control technology in biochemical processes. This work introduces a hybrid ICS-MLSSVM soft-sensor modeling method to realize the online detection of key biochemical variables (cell concentration, substrate concentration, product concentration) of the L-lysine fermentation process. First of all, a multi-output least squares support vector machine regressor (MLSSVM) model is constructed based on the multi-input and multi-output characteristics of L-lysine fermentation process. Then, important parameters ([Formula: see text], [Formula: see text], [Formula: see text]) of MLSSVM model are optimized by using the Improved Cuckoo Search (ICS) optimization algorithm. In the end, the hybrid ICS-MLSSVM soft-sensor model is developed by using optimized model parameter values, and the key biochemical variables of the L-lysine fermentation process are realized online. The simulation results confirm that the proposed regression model can accurately predict the key biochemical variables. Furthermore, the hybrid ICS-MLSSVM soft-sensor model is better than the MLSSVM soft-sensor model based on standard CS (CS-MLSSVM), particle swarm optimization (PSO) algorithm (PSO-MLSSVM) and genetic algorithm (GA-MLSSVM) in prediction accuracy and adaptability.