您的脚本将生成以下命令

cmd /c "C:\Program Files\Microsoft Office\root\Office16\EXCELCNV.EXE" "C:\Users\Tomas\Desktop\fix_excelcnv\Book1.xlsb" "C:\Users\Tomas\Desktop\fix_excelcnv\Book1.xlsx"

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这应该给你给予同样的错误，即使在终端中手动运行（我的猜测是，你运行的命令没有前面的cmd /c，这就是为什么你认为它工作）。
主要的问题在于cmd /c，在cmd /?提供的帮助中有解释：

If /C or /K is specified, then the remainder of the command line after
the switch is processed as a command line, where the following logic is
used to process quote (") characters:

1.  If all of the following conditions are met, then quote characters
    on the command line are preserved:

    - no /S switch
    - exactly two quote characters
    - no special characters between the two quote characters,
      where special is one of: &<>()@^|
    - there are one or more whitespace characters between the
      two quote characters
    - the string between the two quote characters is the name
      of an executable file.

2.  Otherwise, old behavior is to see if the first character is
    a quote character and if so, strip the leading character and
    remove the last quote character on the command line, preserving
    any text after the last quote character.

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因此，如果不需要，可以删除cmd /c，或者将整个命令（3个字符串的序列）包含在字符串中：

cmd /c ""C:\Program Files\Microsoft Office\root\Office16\EXCELCNV.EXE" "C:\Users\Tomas\Desktop\fix_excelcnv\Book1.xlsb" "C:\Users\Tomas\Desktop\fix_excelcnv\Book1.xlsx""

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不要担心它看起来很奇怪，cmd会正确地解析它。
此外，您的命令似乎没有做到您声称的那样-“将xlsb文件转换为xlsx”。
excelcnv.exe没有很好的文档记录，但似乎你缺少了一个-oice标志，所以也许可以尝试以下命令：

cmd /c ""C:\Program Files\Microsoft Office\root\Office16\EXCELCNV.EXE" -oice "C:\Users\Tomas\Desktop\fix_excelcnv\Book1.xlsb" "C:\Users\Tomas\Desktop\fix_excelcnv\Book1.xlsx""

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要在Python中构建此命令，最好按照@picobit的建议使用pathlib，特别是对于更复杂的脚本，但在这里我将提供一个类似于您的代码，添加f和r字符串以提高可读性：

import os

EXCELCNV_PATH = r'"C:\Program Files\Microsoft Office\root\Office16\EXCELCNV.EXE"'
FILE_PATH_STEM = r'C:\Users\Tomas\Desktop\fix_excelcnv\Book1'
XLSB_FILE_PATH = f'"{FILE_PATH_STEM}.xlsb"'
XLSX_FILE_PATH = f'"{FILE_PATH_STEM}.xlsx"'
convert_cmd = f'{EXCELCNV_PATH} -oice {XLSB_FILE_PATH} {XLSX_FILE_PATH}'
cmd = f'cmd /c "{convert_cmd}"'
print(cmd)

os.system(cmd)

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