We explored the relationship between acute ischaemic stroke (IS) early functional outcome and serum levels of homocysteine, vitamin B12, and D in a noninterventional prospective clinical study. We enrolled 50 patients with first-ever IS and performed laboratory tests and functional assessment at three time points: on admission and three and six months after stroke. Modified Rankin Scale (mRS), NIHSS scale, and Barthel index (BI) scores were assessed in all participants by trained examiner blinded to laboratory data. Patients did not receive treatment that might alter laboratory data. Admission NIHSS correlated with homocysteine levels (r = 0.304, p < 0.05), B12 level (r = -0.410, p < 0.01), and vitamin D levels (r = -0.465, p < 0.01). Functional outcome measures (BI and mRS) did not significantly correlate with homocysteine and vitamin D3 levels at 3 and 6 months. However, a positive correlation with vitamin B12 levels was detected for BI both at 3 and 6 months and mRS at 6 months. Higher serum vitamin B12 levels were associated with better functional outcome at follow-up.