Objectives The significance of measuring serum ALP for diagnosing the nature of canine tumors was investigated. Methods A total of 20 canine tumor cases collected from June 2020 to June 2023 were divided into two groups including the patients (experimental group) and the normal (control group). Data were measured and retrospectively analyzed with a micro nano core in the fully automatic animal biochemical instrument based on visual examination and palpation. The site and free state of tumor was characterized with images of X-ray fluoroscopy. Results The content of serum ALP in the experimental group was 4-5 times higher than that in the control group, with significant difference (P<0.01). The results of X-ray fluoroscopy showed that there was a low degree of detachment, unclear depth of adhesion, severe metastasis of tumor, indicating that the tumor is malignant and the prognosis is poor. Conclusions The detection of serum ALP has strong diagnostic value in judging the prognosis of canine tumor patients, and can be further promoted and applied in the clinical practice of small animals.