Fertilization regimes affect the soil microbiological properties of paddy fields. In China, few studies have investigated the influence of long-term fertilizer and combined organic matter and fertilizer application on the soil's microbiological properties. Our objective was to explore the characteristics of soil microbial biomass carbon (SMBC), soil microbial biomass nitrogen (SMBN), and the soil microbial quotient of a paddy field during the early and late rice growth periods under different long-term fertilization regimes in a double cropping rice system. A long-term experiment was established in 1986 in Ningxiang county of Hunan Province, China, and five different fertilizer treatments were applied:(1) no fertilizer input (CK), (2) mineral fertilizer alone (MF), (3) rice residue plus mineral fertilizer (RF), (4) 30% organic matter and 70% mineral fertilizer (OM1), and (5) 60% organic matter and 40% mineral fertilizer (OM2). We analyzed the SMBC, SMBN, and soil microbial quotient of the fields with the above five treatments using the fumigation-extraction and chemical analysis methods. The results showed that after 29 years, the different fertilization regimes significantly affected the soil's chemical and microbial properties. Compared to the CK, the organic matter, total N, and available N, P, and K increased due to the combined application of chemical fertilizer, recycled rice straw, and chicken manure. In paddy fields, the SMBC activity, SMBN, and soil microbial quotient were increased by long-term fertilization regimes wherein applications were carried out during the early and late rice growth periods. Meanwhile, the SMBC activity, SMBN, and soil microbial quotient increased with the development of early and late rice, peaking at the heading stages of early and late rice. These measurements then reached their minimum value at the mature stages of early and late rice. Compared to the CK, the SMBN activity increased by 7.67%, 10.98%, 19.17%, and 21.75% when treated with MF, RF, OM1, or OM2, respectively, at the heading stage of early rice, and increased by 7.27%, 10.41%, 18.18%, and 20.14% when treated with MF, RF, OM1, or OM2 at the heading stage of late rice, respectively. Furthermore, the SMBC activity, SMBN, and the soil microbial quotient were significantly higher when treated with OM1 or OM2 than that in the MF, RF, or CK treatments at the different main growth stages of early and late rice, respectively. In addition, the SMBC activity, SMBN, and soil microbial quotient under different fertilization regimes were ordered as follows:OM2 > OM1 > RF > MF > CK at the main growth stages of early and late rice. The soil fertility was increased by the combined application of mineral fertilizer and organic matter, and the SMBC activity, SMBN, and soil microbial quotient were correlated with combined long-term fertilizer and organic matter application. Therefore, the combined application of chemical fertilizer and recycled rice straw or chicken manure could significantly increase the SMBC activity, SMBN, and soil microbial quotient of paddy fields during early and late rice growth. In addition, the soil microbial properties reflect changes in soil quality, such as the SMBC activity, SMBN, and soil microbial quotient, all of which can be used as biological indicators for the evaluation of soil fertility.