Surface plasmon resonance (SPR) sensing is a real-time detection technique for measuring biomolecular interactions on gold surfaces. This study presents a novel approach using nano-diamonds (NDs) on a gold nano-slit array to obtain an extraordinary transmission (EOT) spectrum for SPR biosensing. We used anti-bovine serum albumin (anti-BSA) to bind NDs for chemical attachment to a gold nano-slit array. The covalently bound NDs shifted the EOT response depending on their concentration. The number of ND-labeled molecules attached to the gold nano-slit array was quantified from the change in the EOT spectrum. The concentration of anti-BSA in the 35 nm ND solution sample was much lower than that in the anti-BSA-only sample (approximately 1/100). With the help of 35 nm NDs, we were able to use a lower concentration of analyte in this system and obtained better signal responses. The responses of anti-BSA-linked NDs had approximately a 10-fold signal enhancement compared to anti-BSA alone. This approach has the advantage of a simple setup and microscale detection area, which makes it suitable for applications in biochip technology.