A mathematical tool used to analyze signals in high-dimensional areas is the biquaternion ambiguity function. While typical ambiguity functions deal with time delay and Doppler frequency, biquaternion ambiguity functions extend the concept to rotations and scaling factors. Biquaternion ambiguity functions offer a more thorough representation of signal ambiguity in these high-dimensional spaces, which can help develop better signal processing algorithms for a range of uses. In this paper, we introduce the notion of biquaternion ambiguity function which is formed from conventional ambiguity function by invoking biquaternion algebra. We first establish the various properties, namely dilation, translation, shift, nonlinearity, boundedness, reconstruction formula, Moyal’s formula, and inversion formula. An interesting relationship between the biquaternion Fourier transform and the biquaternion ambiguity function is also established. Furthermore, we establish the various versions of uncertainty inequalities namely, Pitt’s inequality, sharp Hausdorff–Young inequality, sharp Local uncertainty principle and logarithmic uncertainty principle for biquaternion ambiguity function. Some potential applications are also presented at the end.