A robust electrochemical assay for sorbitol dehydrogenase (SORDH) activity in milk was developed using voltammetry and chronocoulometry at bare and polymer modified transducers. The motivation for the work was to evaluate the potential of SORDH as an early biomarker of bovine pregnancy using milk as sample matrix. SORDH is an enzyme involved in carbohydrate metabolism converting sorbitol, the sugar alcohol form of glucose, into fructose, with NAD+ as a cofactor being simultaneously reduced to NADH. The assay was optimised via direct NADH oxidation on glassy carbon and screen printed carbon electrodes followed by electropolymerisation of 3,4-ethylenedioxythiophene (EDOT) monomer to form an NADH responsive PEDOT surface which operated well in undiluted milk samples. Assay conditions such as incubation time and temperature were optimised resulting in a 3 min assay at 37 °C in the presence of 10 mM NAD+ and 20 mM sorbitol co-substrates, enabling NADH electro oxidation (linear range 0.25–5 mM, sensitivity 9.17 μC cm−2 mM−1 in undiluted milk). SORDH determination followed over the range 0.31–10 U mL−1 in milk samples with sensitivity 5.45 μC cm−2 U−1 mL with LOD 0.0787 U mL−1. The assay was applied to milk sample testing acquired as part of an approved animal study involving control and breeding cycles of dairy cows with focus on analysis at day 19 post artificial insemination. Significant differences between control and pregnant SORDH levels in whole milk animal samples were found (average values 2.57 and 4.07 ng mL−1 respectively), as verified using a commercial SORDH ELISA optical assay. Finally, progesterone monitoring over days 16–21 of the oestrous cycle employed an optical ELISA assay and confirmed maintenance of progesterone levels from day 19 onwards.