Background: High-dose biotin therapy is beneficial in progressive multiple sclerosis (MS). Biotin, as dietary supplement or therapy, may lead to analytical interference in biotin-streptavidin immunoassay. Methods: Seven concentration gradients of biotin solutions were spiked to three different levels of Ca242 serum samples. All samples were tested by CanAg Ca242 ELISA kit to evaluate the interference from biotin. Serum samples with biotin concentration at 1,000 ng/mL were retested after absorption by streptavidin microparticles or direct analysis on the Mindray CL2000i platform. Results: Our study found that CanAg Ca242 is vulnerable to interference when a sample that contains biotin exceeds 15.63 ng/mL. Biotin interference can result in falsely low results in CanAg Ca242. The effect and extent of biotin interference are, to some extent, dependent on the concentration of serum Ca242 and the concentration of biotin. Conclusions: CanAg Ca242 is vulnerable to biotin interference. The laboratory can overcome biotin interference on CanAg Ca242 by using a non-biotin streptavidin method or by absorbing biotin with streptavidin-coated microparticles before testing. Clinicians should use caution in interpreting abnormal results in patients who ingest biotin.