Objective. To explore the clinical significance and value of serum homocysteine (Hcy) and urine 11 dehydrothromboxane B2 (urine 11-DH-TXB2) combined with transferrin-specific peptide (TF-UP) in the diagnosis of stroke. Methods. One hundred stroke patients treated from January 2019 to June 2021 were enrolled in our hospital as the study group. All the patients in the study group met the diagnostic criteria of stroke. The focus of stroke was confirmed by CT or MRI, and the first onset was less than 48 hours. One hundred healthy persons who went through physical examination in our hospital were enrolled as the control group. The comparison was taken to explore the clinical significance and value of Hcy and urine 11-DH-TXB2 combined with TF-UP in the diagnosis of stroke. Results. There exhibited no significant difference in the history of smoking, drinking, and atrial fibrillation (
P
>
0.05
). There were significant differences in systolic blood pressure, diastolic blood pressure, eGFR, history of hypertension, diabetes, and coronary heart disease (
P
<
0.05
). In terms of the levels of Hcy, urine 11-DH-TXB2, and TF-UP, the levels of Hcy and urine 11-DH-TXB2 in the study group were higher compared to the control group, while the level of TF-UP in the study group was lower compared to the control group (
P
<
0.05
). The results of logistic regression analysis indicated that there was a significant correlation between Hcy, urine 11-DH-TXB2, TF-UP, and stroke, and Hcy and urine 11-DH-TXB2 indicated positive correlation with stroke disease, while TF-UP level was negatively correlated with stroke disease (
P
<
0.05
). The levels of Hcy, urine 11-DH-TXB2, and TF-UP were adopted as evaluation indexes to draw ROC curve. The results show that the area under the curve (AUC) of Hcy is 0.760 (95% CI 0.670~0.850). The best critical point was 3342.5 pg/mg Ucr, the sensitivity was 65.6%, and the specificity was 77.1%. The AUC of urine 11-DH-TXB2 was 0.773 (95% CI 0.685~0.861). The best critical point was 3354.44 pg/mg Ucr, the sensitivity was 71.2%, and the specificity was 78.3%. The AUC of TF-UP was 0.735 (95% CI 0.641~0.829). The best critical point was 3365.43 pg/mg Ucr, the sensitivity was 68.4%, and the specificity was 80.5%. If Hcy was detected in combination with other indexes, AUC increased to 0.749 when combined with urine 11-DH-TXB2, and AUC increased to 0.797 when combined with TF-UP. When the three are combined, the AUC can reach 0.836, the sensitivity is 79.1%, and the specificity is 80%. It shows that the combined detection of Hcy, urine 11-DH-TXB2, and TF-UP is of higher diagnostic value. The difference of data exhibited statistically significant (
P
<
0.05
). Conclusion. There is imbalance between Hcy, urine 11-DH-TXB2, and TF-UP in patients with acute stroke. High Hcy, urine 11-DH-TXB2, and low TF-UP are closely related to the occurrence of cerebral infarction. Hcy, urine 11-DH-TXB2, and TF-UP may be the risk factors of stroke and positively correlated with the degree of neurological impairment. Effective monitoring of Hcy and urine 11-DH-TXB2 combined with TF-UP levels and positive intervention measures may effectively prevent the occurrence and development of cerebral infarction, reduce Hcy and urine 11-DH-TXB2, or increase the level of TF-UP, which may provide new ideas for the treatment of cerebrovascular diseases.