Objective To investigate the relationship of the serum levels of homocysteine (Hcy), apolipoprotein H (APOH), and reactive oxygen species (ROS) with intestinal flora imbalance and clinical characteristics in patients with colorectal cancer. Methods Eighty-nine patients with colorectal cancer treated at Daxing Hospital from April 2021 to February 2024 were selected as a study group, including 50 males and 39 females who were (61.45±5.55) years old; 89 healthy people in the same period were selected as a control group, including 52 males and 37 females who were (61.33±5.22) years old. The serum levels of Hcy, APOH, and ROS were detected and compared. The fecal samples of the subjects were collected for bacterial culture, and the number of flora was counted. The numbers of intestinal flora between the study group and the control group were compared. The relationship of the serum levels of Hcy, APOH, and ROS with intestinal flora was analyzed by the Pearson correlation analysis. The clinical characteristics of the patients in the study group were sorted out, and the serum levels of Hcy, APOH, and ROS in the patients with different clinical characteristics were compared. χ2 and t tests were used for the statistical analysis. Results The serum levels of Hcy, APOH, and ROS in the study group were higher than those in the control group [(16.88±5.36) μmol/L vs. (10.54±3.02) μmol/L, (9.66±2.31) mg/L vs. (3.21±1.01) mg/L, and (1 332.58±302.52) U/ml vs. (542.35±30.25) U/ml], with statistical differences (t=9.700, 24.135, and 24.521; all P<0.05). The numbers of Fusobacterium nucleatum, Enterococcus faecalis, and Escherichia coli in the study group were higher than those in the control group [(15.36±2.55) CFU/g vs. (5.06±2.11) CFU/g, (11.13±2.09) CFU/g vs. (4.12±0.99) CFU/g, and (17.16±3.24) CFU/g vs. (5.24±2.11) CFU/g], and the numbers of Bacteroides, Lactobacillus, and Bifidobacterium were lower [(6.87±3.06) CFU/g vs. (16.56±2.44) CFU/g, (8.35±2.41) CFU/g vs. (17.65±4.74) CFU/g, and (6.33±2.05) CFU/g vs. (15.22±4.13) CFU/g], with statistical differences (t=29.539, 28.596, 29.084, 23.358, 16.500, and 18.190; all P<0.05]. The Pearson correlation analysis showed that the serum levels of Hcy, APOH, and ROS were positively correlated with the numbers of Fusobacterium nucleatum, Enterococcus faecalis, and Escherichia coli in the patients (r=0.755, 0.702, and 0.488; r=0.822, 0.733, and 0.577; r=0.801, 0.782, and 0.587; all P<0.05), and negatively with the numbers of Bacteroides, Lactobacillus, and Bifidobacterium (r=-0.632, -0.422, and -0.722; r=-0.664, -0.471, and -0.763; r=-0.678, -0.511, and -0.799; all P<0.05). The serum levels of Hcy, APOH, and ROS in the patients with lymph node metastasis, distant metastasis of tumor, tumor stage III-IV, and poor differentiation were higher than those in the patients without lymph node metastasis, without distant metastasis of tumor, with tumor stage I-II, and with moderate to high differentiation, with statistical differences (all P<0.05). Conclusions The serum levels of Hcy, APOH, and ROS in patients with colorectal cancer are higher than those in healthy people. All the three are related to the intestinal flora imbalance in patients with tumor metastasis, stage, and differentiation.