Bovine respiratory disease (BRD) is a leading cause of mortality and compromised welfare in bovines. It is a polymicrobial syndrome resulting from a complex interplay of viral and bacterial pathogens with environmental factors. Despite the availability of vaccines, incidence and severity in young calves remains unabated. A more precise analysis of host innate immune responses during infection will identify improved diagnostic and prognostic biomarkers for early intervention and targeted treatments to prevent severe disease and loss of production efficiency. Here, we investigate hematological and innate immune responses using standardized ex-vivo whole blood assays in calves diagnosed with BRD. A total of 65 calves were recruited for this study, all between 2–8 weeks of age with 28 diagnosed with BRD by a thoracic ultrasonography score (TUS) and 19 by Wisconsin health score (WHS) and all data compared to 22 healthy controls from the same 9 study farms. Haematology revealed circulating immune cell populations were similar in both TUS positive and WHS positive calves compared to healthy controls. Gene expression analysis of 48 innate immune signalling genes in whole blood stimulated with TLR ligands was completed in a subset of calves. TLR1/2 stimulation with Pam3CSK4 showed a decreased pattern of expression in IL-1 and inflammasome related genes in addition to chemokine genes in calves with BRD. In response to TLR ligands LPS, Pam3CSK4 and R848, protein analysis of supernatant collected from all calves with BRD revealed significantly increased IL-6, but not IL-1β or IL-8, compared to healthy controls. This hyper-induction of IL-6 was observed most significantly in response to TLR1/2 stimulation in TUS positive calves. ROC analysis identified this induced IL-6 response to TLR1/2 stimulation as a potential diagnostic for BRD with a 74% true positive and 5% false positive detection rate for an IL-6 concentration >1780pg/mL. Overall, these results show altered immune responses specifically upon TLR1/2 activation is associated with BRD pathology which may contribute to disease progression. We have also identified induced IL-6 as a potentially informative biomarker for improved early intervention strategies for BRD.