© 2017 The Author(s). Background: Early signs of canine transitional cell carcinoma (TCC) are frequently assumed to be caused by other lower urinary tract diseases (LUTD) such as urinary tract infections, resulting in late diagnosis of TCC which could be fatal. The development of a non-invasive clinical test for TCC could dramatically reduce mortality. To determine whether microRNAs (miRNAs) can be used as non-invasive diagnostic biomarkers, we assessed miRNA expression in blood and/or urine from dogs with clinically normal bladders (n = 28), LUTD (n = 25), and TCC (n = 17). Expression levels of 5 miRNA associated with TCC pathophysiology (miR-34a, let-7c, miR-16, miR-103b, and miR-106b) were assessed by quantitative real-time PCR. Results: Statistical analyses using ranked ANOVA identified significant differences in miR-103b and miR-16 levels between urine samples from LUTD and TCC patients (miR-103b, p = 0.002; and miR-16, p = 0.016). No statistically significant differences in miRNA levels were observed between blood samples from LUTD versus TCC patients. Expression levels of miR-34a trended with miR-16, let-7c, and miR-103b levels in individual normal urine samples, however, this coordination was completely lost in TCC urine samples. In contrast, co-ordination of miR-34a, miR-16, let-7c, and miR-103b expression levels was maintained in blood samples from TCC patients. Conclusions: Our combined data indicate a potential role for miR-103b and miR-16 as diagnostic urine biomarkers for TCC, and that further investigation of miR-103b and miR-16 in the dysregulation of coordinated miRNA expression in bladder carcinogenesis is warranted.