Second generation ethanol faces challenges before profitable implementation. Biomass hydrolysis is one of the bottlenecks, especially when this process occurs at high solids loading and with enzymatic catalysts. Under this setting, kinetic modeling and reaction monitoring are hindered due to the conditions of the medium, while increasing the mixing power. An algorithm that addresses these challenges might improve the reactor performance. In this work, a soft sensor that is based on agitation power measurements that uses an Artificial Neural Network (ANN) as an internal model is proposed in order to predict free carbohydrates concentrations. The developed soft sensor is used in a Moving Horizon Estimator (MHE) algorithm to improve the prediction of state variables during biomass hydrolysis. The algorithm is developed and used for batch and fed-batch hydrolysis experimental runs. An alteration of the classical MHE is proposed for improving prediction, using a novel fuzzy rule to alter the filter weights online. This alteration improved the prediction when compared to the original MHE in both training data sets (tracking error decreased 13%) and in test data sets, where the error reduction obtained is 44%.