In this paper, the problem of sampled-data observer-based anti-windup (AW) control for singularly perturbed systems with actuator saturation is considered. A sampled-data observer-based AW controller consisting of a sampled-data observer, controller and AW compensator is proposed for the first time. Based on an ε-dependent Lyapunov-Krasovskii functional and linear matrix inequalities, the three components of sampled-data observer-based AW controller are designed simultaneously, which can reduce the conservatism. Furthermore, a convex optimization algorithm is formulated to obtain a desired stability bound and enlarge the basin of attraction at the same time. Finally, examples are provided to demonstrate the effectiveness and merits of the obtained results.