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A new robust on-line fault diagnosis method based on least squares estimate for nonlinear difference-algebraic systems (DAS) with uncertainties is proposed. Based on the known nominal model of the DAS, this method firstly constructs an auxiliary system consisting of a difference equation and an algebraic equation, then, based on the relationship between the state deviation and the faults in the difference equation and the relationship between the algebraic variable deviation and the faults in algebraic equation, it identifies the faults on-line through least squares estimate. This method can not only detect, isolate and identify faults for DAS, but also give the upper bound of the error of fault identification. The simulation results indicate that it can give satisfactory diagnostic results for both abrupt and incipient faults.