In order to study the influence of various factors on the alfalfa conditioning performance, a set of the measurement and control system for alfalfa conditioning test bench was built with the LabVIEW software on the basis of the existing alfalfa conditioning test bench. The measurement and control system was mainly composed of four parts, including a motor control and monitoring system, a data acquisition system, a conditioning gap regulating system and a PC system. By the measurement and control system with the data acquisition frequency of 1kHz, the conditioning rotate speed can be adjusted continuously in the range from 350r/min to 1350r/min, and the conditioning gap between both rubber rollers can be controlled accurately between 2mm and 4mm. The curves, such as the torque and the speed of the fixed roller, the power of the motor, the pressure of the hydraulic cylinder used for regulating the gap between the rollers, were displayed and saved in real time on the front panel of the measurement and control system. A three-factor three-level response surface test with the Box-Behnken test method was carried out using alfalfa with the alfalfa conditioning test bench equipped with a measurement and control system. The results showed that the measurement and control system could achieve precise control of the alfalfa conditioning test bench and real-time accurate data collection. By the analysis of the test data, the quadratic regression models of the unit energy consumption, the alfalfa flattening rate and the flattening loss rate were established, respectively, the optimal solution of the working parameters of the alfalfa conditioning test bench were achieved, that was, the rotate speed of the conditioning rollers was 775r/min, and the gap of conditioning rollers was 3.3mm, and the feeding rate of the per unit working length of the conditioning rollers was 2.77kg/(m·s). Meanwhile, the optimal solutions of the conditioning qualities of alfalfa under the optimal working parameters conditions were achieved, respectively, that was, the unit energy consumption was 909.25J/kg, the flattening rate of alfalfa was 96.67%, and the flattening loss rate was 1.67%. The working parameters of the alfalfa conditioning test bench were set as the optimal solution parameters of the models listed above, the confirmation tests with the alfalfa were carried out. The results showed that the measurement values of the conditioning quality parameters of the alfalfa under the optimal working parameters conditions were consistent with the optimization results, that was, the unit energy consumption, the flattening rate and the flattening loss rate of alfalfa were 931.42J/kg, 94.33% and 1.65%, respectively, and the relative errors between the solution optimization values of the models and the measured values of the confirmation tests were less than 3%. The results showed that the measurement and control system provided a reliable technical support for the alfalfa conditioning test, and provided a data reference and theoretical basis for the design of alfalfa conditioning mechanism and the selection of working parameters.