In this study, the greenhouse soils under tomato-cucumber rotation with four treatments (1/2 chemical fertilizer N+1/2 bio-organic fertilizer N, COF; all bio-organic fertilizer N, OF; all chemical fertilizer N, CF; no fertilizer treatment, CK) were taken as the research objects to explore the effect of bio-organic fertilizer combined with chemical fertilizer on nutrient content, aggregate distribution and stability of greenhouse soil. The results showed that compared with CF, the application of bio-organic fertilizer increased soil organic carbon, total nitrogen, total phosphorus, total potassium, available phosphorus, available potassium contents and soil pH by 9.61%-54.28%, 7.38%-35.45%, 31.86%-98.53%, 40.88%-96.40%, 3.02%-15.99%, 0.96%-18.23% and 0.73%-7.03%, respectively. The application of bio-organic fertilizer alone or in combination with chemical fertilizer increased the proportion of soil macroaggregate (>0.25 mm) while decreased the proportion of soil microaggregate (<0.25 mm), significantly increased (P<0.05) the mean mass diameter (MWD), geometric mean diameter (GMD) and aggregate content>0.25 mm (R_0.25) of soil aggregates, and the stability of soil aggregates was increased with the year increase of application of bio-organic fertilizer. MWD, GMD and R_0.25 were significantly positively correlated with the mass fractions of aggregates >2 mm and 0.25-2 mm (P<0.01), while negatively correlated with that of aggregates <0.053 mm (P<0.01) and 0.053-0.25 mm (P<0.05). In conclusion, bio-organic fertilizer is more beneficial to improve soil nutrient contents, the number of large aggregates and the stability of aggregates than chemical fertilizer.