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High-accuracy servo control of pneumatic systems requires nonlinear model-based control techniques based on full-state knowledge of the system. For the consideration of cost or light-weight design, non-linear pressure observers in place of pressure sensors were constructed to acquire the pressure states in the chambers of the pneumatic cylinder. Firstly, the global stability of the proposed pressure observer was demonstrated based on Lyapunov-stability theory. Secondly, the pressure observer’s polytropic exponent was analyzed and determined experimentally. Moreover, to check the fast convergence of the proposed pressure observer, a set of experiments for tracking a 0.5 Hz sinusoidal trajectory were performed under condition that the initial states of both chambers should be pressurized and the initial values of the observed pressures were intentionally set different from the actual values. Furthermore, to verify the load-independent of the presented pressure observer, the valve-controlled pneumatic cylinder system acted upon by a variable-stiffness loading force was adopted to carry out pneumatic servo control. A three-way on-off solenoid value was configured as two-way valve. Thus, four two-way on-off solenoid valves controlled with pulse width modulation (PWM) in lieu of one or two proportional valves were employed to govern the pneumatic cylinder for achieving the pneumatic servo control. Finally, extensive experiments validated the effectiveness and feasibility of the proposed pressure observer for high-accuracy pneumatic servo control of the valve-controlled pneumatic cylinder system.