In biometric systems, reference threshold is defined as a value that can decide the authenticity of a person. Authenticity means whether the person is genuine or intruder. The statistical calculation of various values like reference threshold, FAR (False Acceptance Rate), FRR (False Rejection Rate) is required for real-time automated biometric authentication system because the measurement of biometric features is statistical values. In this paper, the need of reference threshold, how reference threshold value is calculated is explained mathematically. Various factors on which reference threshold value depends are discussed.