As a nano-Newton level force sensor, an AFM probe with a protein-modified tip could be used to investigate the antifouling properties of different ultrafiltration membranes. In this paper, the spring constants of the cantilevers of AFM probes were calibrated with a SI-traceable measurement system consisting of a precision balance, an optical lever and nano-positioning stages. The calibration system for the measured AFM cantilevers has less than 3% uncertainty, highly narrowing the large range given by the distributers. Thus, the adhesion forces between the AFM probes with different immobilized proteins and various ultrafiltration membranes could be compared with each other using force curves. The antifouling universality of a specific membrane (polyvinyl formal, PVF) towards multiple proteins (bovine serum albumin, lysozyme, catalase and fibrinogen) could be studied comprehensively. The results reveal that the antifouling property of membranes becomes better through blending F127 with PVF for all four foulants, so the antifouling universality of the membrane was improved effectively. The membrane with 60% F127 addition had the best antifouling properties. Comparison experiments of protein absorption and water contact angle have also been done. As the F127 additive varied from 0% to 60%, the amount of protein adsorption on the surface of the membranes decreased significantly and the water contact angle decreased from 58.6° to 47.1°, which indicated that the blend membranes had better antifouling universality. The experimental results agreed with AFM force curve experiments very well.