Doxorubicin (DOX) is a widely employed tumor therapy, yet its substantial toxic side effects pose a considerable challenge. Bletilla striata has demonstrated efficacy in preventing and treating these toxic side effects in clinical practice, with polysaccharides identified as the principal active component. In the present study, 16 fractions of B. striata polysaccharides (BsPs) were extracted using diverse methods, including hot-water extraction (HWE), ultrasonic-assisted extraction (UAE), enzyme-assisted extraction (EAE), dilute acid-water extraction (ACWE), and dilute alkali-water extraction (ALWE). These extractions were subsequently precipitated at final ethanol concentrations of 80% and 95%, respectively. The investigation encompassed yields, total carbohydrate content (TCC), total protein content (TPC), preliminary structural properties, and anti-DOX myocardial cytotoxic activity. Results indicated that the extraction method significantly influenced the physicochemical properties, associated functional properties, and anti-DOX myocardial cytotoxic activity of BsPs. HWE and UAE yielded higher BsPs quantities. The relative molecular weight (RMW) distribution of BsPs differed notably between HWE or UAE, EAE, ACWE, and ALWE. The RMW of primary BsPs obtained from HWE and UAE (1.9 × 107–1.7 × 107 Da) exceeded that from EAE (7.5 × 103–2.8 × 104 Da) and ALWE (5.1 × 104–1.7 × 104 Da), with smaller molecular weights primarily precipitated by higher ethanol concentrations. BsPs were composed of Man and Glu, with partial fractions containing small amounts of Gal or Ara, displaying varying molar ratios. Notably, BsPs from ACWE exhibited the most significant structural differences, lacking 1,4-α-D-Glcp and a triple-helical structure. Furthermore, BsPs obtained from HWE, UAE, and EAE demonstrated heightened anti-DOX myocardial cytotoxic activity compared to other methods. This study underscored the influence of extraction methods on BsPs’ structure and myocardial protective activity, offering a foundation for exploring structural diversity and employing specific extraction methods to extract polysaccharides with robust myocardial protective properties efficiently.