Real-time pH measurement and regulation of microliter-scale liquid samples are critical for microfluidic biochemical applications. However, traditional magnetic stirring for macroscopic systems is not applicable under such microscale conditions. This work develops an ultrasound-assisted screen-printed electrode (SPE) pH sensor integrated with a 3D-printed ultrasonic reflection cell and piezoelectric excitation unit. Ultrasound-induced acoustic streaming accelerates mass transfer inside microliter-scale samples. Upon acid addition, the ultrasonic-assisted system reaches potentiometric equilibrium far more rapidly than the static non-ultrasonic control. Optimized parameters are 30 s ultrasonic duration and 5 Vpp input amplitude. The sensor shows linearity from pH 3.04 to 4.15 with R² = 0.9919. Phenolphthalein visualization directly confirms ultrasound-enhanced molecular diffusion. Its mass transfer efficiency matches traditional magnetic stirring, and stable fast equilibrium is realized in complex cell culture medium. This acoustofluidic micromixing strategy offers a simple integrated route for pH monitoring and the regulation of micro-volume biological samples.