We have developed a simple technique for detecting protein in raw blood samples using size-exclusion surface plasmon resonance (SPR) sensing. We designed a sensor chip consisting of a microslit array fabricated on a flat gold surface with gaps that were smaller in size than a mammalian red blood cell, which was designed for use in various Kretschmann-type SPR instruments. Blood cells were blocked by the microwalls, whereas the serum reached the SPR sensing field, enabling cell filtration and immediate protein detection in a one-step operation that required only the dispensing of a blood sample onto the chip. We evaluated the influence of the chip structure on SPR characteristics, and demonstrated using a whole blood sample from horse that IgG can be detected using the chip within 100 s of application.