On-line estimation of unmeasurable biological variables is important in fermentation processes, directly influencing the optimal control performance of the fermentation system as well as the quality and yield of the targeted product. In this study, a novel strategy for state estimation of fed-batch fermentation process is proposed. By combining a simple and reliable mechanistic dynamic model with the sample-based regressive measurement model, a state space model is developed. An improved algorithm, swarm energy conservation particle swarm optimization (SECPSO), is presented for the parameter identification in the mechanistic model, and the support vector machines (SVM) method is adopted to establish the nonlinear measurement model. The unscented Kalman filter (UKF) is designed for the state space model to reduce the disturbances of the noises in the fermentation process. The proposed on-line estimation method is demonstrated by the simulation experiments of a penicillin fed-batch fermentation process.